Rapid assay for simultaneous detection and differentiation of antibodies to HIV groups

ABSTRACT

A method of performing a rapid assay for the simultaneous detection and differentiation of the analytes HIV-1 group M. HIV-1 group O and HIV-2 utilizing a sequence specific polypeptide of each analyte as capture reagents. An analytical. device also is provided for performing the method which includes these capture reagents. Also provided is a test kit which includes the analytical device which further can include a positive and negative control.

BACKGROUND OF THE INVENTION

This invention relates generally to immunoassays, and more particularly,relates to an immunoassay useful for detecting and differentiatingantibodies to Human Immunodeficiency Virus Type 1 (HIV-1) group M, HIV-1group O and Human Immunodeficiency Virus Type 2 (HIV-2) in test sampleswith a rapid turn-around time.

Currently, there are two major phylogenetic groups of HIV-1 designatedas groups "M" and "O." G. Meyers et al., Human Retroviruses and AIDS1995, Los Alamos National Laboratory, Los Alamos, N.Mex. (1995). HIV-1group M isolates further have been divided into subgroups (A to J) thatare phylogenetically approximately equidistant from each other. Group Misolates predominate worldwide. The earliest reports about the sequenceof HIV-1 group O viruses indicated that these viruses were as closelyrelated to a chimpanzee virus as to other HIV-1 subgroups. See, forexample, L. G. Gurtler et al., J. Virology 68: 1581-1585 (1994); M.Vanden Haesevelde et al., J. Virology 68: 1586-1596 (1994); De Leys etal., J. Virology 64: 1207-1216 (1990); DeLeys et al., U.S. Pat. No.5,304,466; L. G. Gurtler et al., European Patent Publication No.0591914A2. The group O sequences are the most divergent of the HIV-1sequences described to date. Although HIV-1 group O strains are endemicto west central Africa (Cameroon, Equatorial Guinea, Gabon, andNigeria), patients infected with group O isolates now have beenidentified in Belgium, France, Germany, Spain and the United States.See, for example, R. DeLeys et al., supra; P. Charneau et al., Virology205:247-253 (1994); I. Loussert-Ajaka et al., J. Virology 69:5640-5649(1995); H. Hampl et al., Infection 23:369-370 (1995); A. Mas et al.,AIDS Res. Hum. Retroviruses 12:1647-1649 (1996); M. A. Rayfield et al.,Emerging Infectious Diseases 2:209-212 (1996), and M. Peeters et al.,AIDS 11:493-498 (1997).

HIV-1 group M serology is characterized in large part by the amino acidsequences of the expressed viral proteins (antigens), particularly thosecomprising the core and envelope (env) regions. These antigens arestructurally and functionally similar, but have divergent amino acidsequences that elicit antibody responses which are specific for theparticular antigen.

One of the key serological targets for detection of HIV-1 infection isthe 41,000 molecular weight transmembrane protein (TMP), glycoprotein(gp)41. gp41 is a highly immunogenic protein which elicits a strong andsustained antibody response in individuals considered seropositive forHIV. Antibodies to this protein are among the first to appear atseroconversion. The immune response to gp41 apparently remainsrelatively strong throughout the course of the disease, as evidenced bythe near universal presence of anti-gp41 antibodies in asymptomatic aswell as clinical stages of AIDS. A significant proportion of theantibody response to gp41 is directed toward a well-characterizedimmunodominant region (IDR) within gp41.

HIV-2 infections have been identified in humans outside of the initialendemic area of West Africa, and have been reported in Europeans whohave lived in West Africa or those who have had sexual relations withindividuals from this region, homosexuals with sexual partners from theendemic area, and others. Cases of AIDS due to HIV Type 2 (HIV-2) nowhave been documented world-wide. See, for example, A. G. Saimot et al.,Lancet i:688 (1987); M. A. Rey et al., Lancet i:388-389 (1987); A.Werner et al., Lancet i:868-869 (1987); G. Brucker et al., Lancet i:223(1987); K. Marquart et al., AIDS 2:141 (1988); CDC, MMWR 37:33-35(1987); Anonymous, Nature 332:295 (1988).

Serologic studies indicate that while HIV-1 and HIV-2 share multiplecommon epitopes in their core antigens, the envelope glycoproteins ofthese two viruses are much less cross-reactive. F. Clavel, AIDS1:135-140 (1987). This limited cross-reactivity of the envelope antigensis believed to explain why currently available serologic assays forHIV-1 may fail to react with certain sera from individuals with antibodyto HIV-2. F. Denis et al., J. Clin. Micro. 26:1000-1004 (1988). Recentlyissued U.S. Pat. No. 5,055,391 maps the HIV-2 genome and provides assaysto detect the virus.

Concerns have arisen regarding the capability of currently availableimmunoassays for the detection of antibody to HIV-1 (group M) and/orHIV-2 to detect the presence of antibody to HIV-1 group O. I.Loussert-Ajaka et al., Lancet 343:1393-1394 (1994); C. A. Schable etal., Lancet 344:1333-1334 (1994); L. Gurtler et al., J. Virol. Methods51:177-184 (1995). Compounding the problem of analyzing whether theseimmunoassays are capable of detecting group O is the limitedavailability of sera samples from patients who are infected with and/orhave antibody to HIV-1 group O isolates. To date, few patients have beendiagnosed with infection to HIV-1 group O isolates outside of westCentral Africa, leading researchers to screen patients in west centralAfrican countries for the virus. Screening procedures in west centralAfrica have been hampered both by the time necessary to perform theseassays as well as the equipment required to do so. Conventional bindingassays available for detecting antibodies to HIV-1 group M, HIV-1 groupO and HIV-2 usually take about two to four or more hours to reach aresult. These assays further involve utilizing equipment includingincubators and label reading devices that require electricity in orderto operate. These assays incorporate specific binding members, usuallyantibody and antigen immunoreactants, wherein one member of the specificbinding pair is labeled with a signal-generating compound (e.g., anantibody labeled with an enzyme, a fluorescent compound, achemiluminescent compound, a radioactive isotope, a direct visual label,etc.). The test sample suspected of containing the analyte can be mixedwith a labeled reagent, e.g., labeled anti-analyte antibody, andincubated for a time and under conditions sufficient for theimmunoreaction to occur. The reaction mixture is subsequently analyzedto detect either that label which is associated with the analyte/labeledreagent complex (bound labeled reagent) or that label which is notcomplexed with analyte (free labeled reagent). The presence and/oramount of an analyte is indicated by the analyte's capacity to bind to alabeled reagent and binding member, which usually is immobilized or aninsoluble complementary binding member.

There are situations and places in which the period of time usuallyrequired to perform these assays and report results is too long (i.e.,two to four hours), or the equipment and/or electricity necessary to runthe assay is not available. In such situations, a preferable test shouldbe inexpensive, require little or no equipment, and provide a result fora screening assay in as little time as five minutes.

The use of reagent-impregnated teststrips in specific binding assays iswell-known. See, for example, Deutsch et al., U.S. Pat. No. 4,361,537and Brown et al., U.S. Pat. No. 5,160,701. In such procedures, a testsample is applied to one portion of the teststrip and is allowed tomigrate or wick through the strip material. Thus, the analyte to bedetected or measured passes through or along the material, possibly withthe aid of an eluting solvent which can be the test sample itself or aseparately added solution. The analyte migrates into or through acapture or detection zone on the teststrip, wherein a complementarybinding member to the analyte is immobilized. The extent to which theanalyte becomes bound in the detection zone can be determined with theaid of the labeled reagent which also can be incorporated into theteststrip or which can be applied separately.

In general, teststrips involve a material capable of transporting asolution by capillary action, i.e., a wicking or chromatographic actionas exemplified in Gordon et al., U.S. Pat. No. 4,956,302. Differentareas or zones in the teststrip contain the assay reagents needed toproduce a detectable signal as the analyte is transported to or throughsuch zones. The device is suitable both for chemical assays and bindingassays and uses a developer solution to transport analyte along thestrip. Also, to verify the stability and the efficacy of the assayreagents needed to produce the detectable signal, existing assaystypically require at least that one or more strips from eachmanufacturing lot be separately assayed for both positive and negativecontrols.

Assay systems developed for the separate or concurrent detection ofantibodies to HIV-1 group M, and/or HIV-1 group O and/or HIV-2 thereforemust contain reagents which are useful for determining the specificpresence of antibody to any or all of the viruses in a test sample whiledifferentiating between them. The need therefore exists for reagentscapable of reacting only with antibody to HIV group M, HIV group O andHIV-2, which reagents either exhibit no cross-reactivity or limitedcross-reactivity with each other. It also would be beneficial to providea disposable assay device which could incorporate these reagents and beused for screening individuals and providing results in a short amountof time.

SUMMARY OF THE INVENTION

The present invention provides a method for simultaneously detecting anddifferentiating between analytes comprising antibodies to HIV-1 group O,HIV-1 group M and HIV-2 in a test sample. The method comprises (a)contacting the test sample with an analytical device having a strip witha proximal end and a distal end, wherein the test sample moves from theproximal end to about the distal end by capillary action, and whereinthe strip contains at least one immobilized capture reagent per analyte,for a time and under conditions sufficient to form capturereagent/analyte complexes by the binding of the analyte and the capturereagent; and (b) determining the presence of the analyte(s) by detectinga visible color change at the capture reagent site on the strip, whereinthe capture reagent for HIV-1 group O comprises a polypeptide selectedfrom the group consisting of SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52and SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60, the capture reagentfor HIV-1 group M comprises a polypeptide SEQ ID NO: 56, and the capturereagent for HIV-2 comprises a polypeptide SEQ ID NO: 55. Preferably, thepolypeptide capture reagent is prepared by recombinant technology,although it is contemplated that a purified protein (polypeptide) or asynthetic peptide may be utilized. The immobilized capture reagent canbe configured as a letter, number, icon, or symbol. Further, the methodcomprises an indicator reagent contained within the strip in a situsbetween the proximal end and the immobilized patient capture reagent.The indicator reagent comprises a signal generating compound, whichcompound is selected from the group consisting of a chromogen, acatalyst, a luminescent compound, a chemiluminescent compound, aradioactive element and a direct visual label. Preferably, the indicatorreagent comprises a direct visual label selected from the groupconsisting of colloidal metallic particles, colloidal non-metallicparticles, dyed or colored particles, and liposomes. The indicatorreagent further comprises selenium as a non-metallic particle. The testsample preferably is a body fluid. The body fluid is selected from thegroup consisting of whole blood, plasma, serum, urine, and saliva.

The present invention further provides an analytical device forsimultaneous detecting and differentiating between HIV-1 group O, HIV-1group M and HIV-2 in a test sample, comprising a strip with a proximalend and a distal end, wherein the test sample is capable of moving fromthe proximal end to about the distal end by capillary action, andwherein the strip contains at least one immobilized capture reagent peranalyte, for binding of the analyte and the capture reagent; and whereinthe capture reagent for HIV-1 group O comprises a polypeptide sequenceselected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 50, SEQID NO: 52, and SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60, saidcapture reagent for HIV-1 group M comprises SEQ ID NO: 56, and saidcapture reagent for HIV-2 comprises SEQ ID NO: 55. The polypeptidepreferably is produced by recombinant technology, although it iscontemplated that purified protein (polypeptide) and synthetic peptidescan be used. The analytical device further comprises an immobilizedcapture reagent that is configured as a letter, number, icon, or symbol.Further, the analytical device comprises an indicator reagent that iscontained within the strip in a situs between the proximal end and theimmobilized patient capture reagent. The indicator reagent comprises asignal generating compound which compound is selected from the groupconsisting of a chromogen, a catalyst, a luminescent compound, achemiluminescent compound, a radioactive element, and a direct visuallabel. Preferably, the indicator reagent comprises a direct visual labelselected from the group consisting of colloidal metallic particles,colloidal non-metallic particles, dyed or colored particles, andliposomes. The test sample preferably is a body fluid. The body fluid isselected from the group consisting of whole blood, plasma, serum, urine,and saliva.

In addition, the present invention provides a test kit for use inspecific binding assays. The test kit comprises an analytical device fordetermining the presence or amount of HIV-1 group O, HIV-1 group M andHIV-2 specific antibodies in a test sample, and further comprises astrip having a proximal end and a distal end, wherein the test sample iscapable of moving from the proximal end to about the distal end bycapillary action, and wherein the strip contains an immobilized capturereagent that binds to a member selected from the group consisting of theanalyte, an ancillary specific binding member and an indicator reagent.The capture reagent for HIV-1 group O comprises a polypeptide selectedfrom the group consisting of SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:52, and SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60, said capturereagent for HIV-1 group M comprises SEQ ID NO: 56, and said capturereagent for HIV-2 comprises SEQ ID NO: 55. The polypeptide preferably isproduced by recombinant technology. It is contemplated that a purifiedprotein or a synthetic peptide also may be used. The indicator reagentcomprises a signal generating compound which compound is selected fromthe group consisting of a chromogen, a catalyst, a luminescent compound,a chemiluminescent compound, a radioactive element and a direct visuallabel. Preferably, the indicator reagent comprises a direct visual labelselected from the group consisting of colloidal metallic particles,colloidal non-metallic particles, dyed or colored particles, andliposomes. The test kit further comprises a positive reagent control anda negative reagent control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the deduced amino acid sequence of the env protein fromthe HIV-1 group O isolate HAM112 (SEQ ID NO: 61).

FIG. 2 depicts the strategy used to generate synthetic HIV-1 group O envgp120/gp41 gene constructs, wherein the pGO-8 insert=Osyn-5' toOsyn-P3'; pGO-9 insert=Osyn-5' to Osyn-03'; pGO-11 insert=Osyn-5' toOsyn-M; and wherein H=the hydrophobic region of HIV-1 group O, deletedas shown.

FIGS. 3A through 3D show a diagrammatic representation of the stepsinvolved in construction of pGO-9PL/DH5α and pGO-9CKS/XL1.

FIGS. 4A through 4G show a diagrammatic representation of the stepsinvolved in construction of pGO-11PL/DH5α and pGO-11CKS/XL1.

FIG. 5 illustrates the amino acid sequence of the pGO-8PL recombinantprotein (SEQ ID NO: 58).

FIG. 6 shows the amino acid sequence of the pGO-8CKS recombinant protein(SEQ ID NO: 60).

FIG. 7 illustrates the amino acid sequence of the pGO-9PL recombinantprotein (SEQ ID NO: 48).

FIG. 8 shows the amino sequence of the pGO-9CKS recombinant protein (SEQID NO: 50).

FIG. 9 illustrates the amino acid sequence of the pGO-11PL recombinantprotein (SEQ ID NO: 52).

FIG. 10 shows the amino sequence of the pGO-11CKS recombinant protein(SEQ ID NO: 54).

FIG. 11 illustrates the amino acid sequence of the pHIV-210 recombinantprotein (SEQ ID NO: 55).

FIG. 12 is a front plan view of the test device utilized for the presentinvention.

FIG. 13 is a cross-section view of the test device shown in FIG. 12,taken along lines (20)-(22) of FIG. 12.

FIG. 14 is a photograph of the results obtained in four test devices of(from left to right) two negative serum samples (two test devices to theleft) and two negative whole blood test samples (two test devices to theright) spiked with a negative control in the assay of the invention.

FIG. 15 is a photograph of ten test devices and shows the resultsobtained testing (from left to right) five HIV-1 group M sera (five testdevices to the left) and five whole blood samples (five test devices tothe right) spiked with the HIV-1 group M positive sera.

FIG. 16 is a photograph of four test devices showing the resultsobtained when testing (from left to right) two confirmed positive HIV-1group O sera (two test devices to the left) and two whole blood testsamples spiked with HIV-1 group O sera (two test devices to the right).

FIG. 17 is a photograph of ten test devices showing the results obtainedwith (from left to right) five HIV-2 confirmed positive sera (five testdevices to the left) and whole blood spiked with HIV-2 sera (five testdevices to the right).

FIG. 18 is a photograph of four test devices, in which (from left toright) a negative test sample, an HIV-1 group M positive test sample, anHIV-1 group O positive test sample, and an HIV-2 positive test samplewere tested individually.

DETAILED DESCRIPTION OF THE INVENTION

The ability to screen for HIV-1 group M, HIV-1 group O and HIV-2 in lesstime than conventional assays is a required feature in situations inwhich quick results are necessary for patient counseling and treatment.Such a screening assay must be able to provide a similar degree ofsensitivity and specificity as the conventional screening assays, but ina much shorter period of time. The present invention provides such anassay and is described hereinbelow.

The following terms have the following meanings unless otherwise noted:

The term "test sample" refers to a component of an individual's bodywhich is the source of the analyte (such as, antibodies of interest orantigens of interest).

These components are well-known in the art. The test sample can be useddirectly as obtained from the source or after pretreatment so as tomodify its character. These test samples include biological sampleswhich can be tested by the methods described herein and include humanand animal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; and biological fluids such ascell culture supernatants; fixed tissue specimens; and fixed cellspecimens. The test sample can be pretreated prior to use, such aspreparing plasma from blood, diluting viscous fluids, or the like;methods of treatment can involve extraction, filtration, distillation,concentration, inactivation of interfering components, and the additionof reagents. Such pretreatment also can include the modification of asolid material suspected of containing the analyte to form a liquidmedium or to release the analyte.

"Analyte," as used herein, is the substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as, anantibody), or for which a specific binding member can be prepared. Thus,an analyte is a substance that can bind to one or more specific bindingmembers in an assay. "Analyte" also includes any antigenic substances,haptens, antibodies, and combinations thereof. As a member of a specificbinding pair, the analyte can be detected by means of naturallyoccurring specific binding partners (pairs) for example, but not limitedto, the use of intrinsic factor protein as a member of a specificbinding pair for the determination of Vitamin B12, the use offolate-binding protein to determine folic acid, or the use of a lectinas a member of a specific binding pair for the determination of acarbohydrate. The analyte includes any antigenic substances such as butnot limited to a protein, a peptide, an amino acid, a nucleotide target,and the like, haptens, antibodies, macromolecules and combinationsthereof.

"Analyte-analog" refers to a substance which cross-reacts with theanalyte-specific binding member, although it may do so to a greater or alesser extent than does the analyte itself. The analyte-analog caninclude a modified analyte as well as a fragmented or synthetic portionof the analyte molecule, so long as the analyte-analog has at least oneepitopic site in common with the analyte of interest. An example of ananalyte-analog is a synthetic peptide sequence which duplicates at leastone epitope of the whole molecule analyte so that the analyte-analog canbind to the analyte-specific binding member.

The present invention provides assays which utilize specific bindingmembers. A "specific binding member," as used herein, is a member of aspecific binding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude for example without limitation biotin and avidin, carbohydratesand lectins, complementary nucleotide sequences, effector and receptormolecules, cofactors and enzymes, enzyme inhibitors and enzymes, and thelike. In addition, other specific binding pairs include, as exampleswithout limitation, complementary peptide sequences, a peptide sequenceand an antibody specific for the sequence or the entire protein,polymeric acids and bases, dyes and protein binders, peptides andspecific protein binders (for example, ribonuclease, S-peptide andribonuclease S-protein). Furthermore, specific binding pairs can includemembers that are analogs of the original specific binding members, forexample, an analyte-analog. The specific binding pair member can includea protein, a peptide, an amino acid, a nucleotide target, and the like.Immunoreactive specific binding members include antigens, antigenfragments, antibodies and antibody fragments, both monoclonal andpolyclonal, and complexes thereof, including those formed by recombinantDNA molecules, folate-binding protein to determine folic acid, or theuse of a lectin as a member of a specific binding pair for thedetermination of a carbohydrate.

The term "hapten", as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

The "indicator reagent" which also is referred to as a "labeled reagent"comprises a "signal generating compound" ("label") which is capable ofgenerating and generates a measurable signal detectable by externalmeans conjugated (attached) to a specific binding member for HIV. Inaddition to being an antibody member of a specific binding pair for HIV,the indicator reagent also can be a member of any specific binding pair,including either hapten-anti-hapten systems such as biotin oranti-biotin, avidin or biotin, a carbohydrate or a lectin, acomplementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to HIV as in a sandwich assay, to the capture reagent as in acompetitive assay, or to the ancillary specific binding member as in anindirect assay. The attachment of the signal generating compound and thespecific binding member may be by covalent or non-covalent binding, butthe method of attachment is not critical to the present invention. Thelabel allows the indicator reagent to produce a detectable signal thatis directly or indirectly related to the amount of analyte in the testsample. The specific binding pair member component of the indicatorreagent is selected to directly bind to the analyte or to indirectlybind to the analyte by means of an ancillary specific binding member.The labeled reagent can be incorporated in the test device, it can becombined with the test sample to form a test solution, it can be addedto the device separately from the test sample or it can be predepositedor reversibly immobilized at the capture site. In addition, the bindingmember may be labeled before or during the performance of the assay bymeans of a suitable attachment method.

The various "signal generating compounds" ("labels") contemplatedinclude chromogens, catalysts such as enzymes, luminescent compoundssuch as fluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements, and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase, and the like.Examples of direct visual labels include colloidal metallic particlessuch as gold, colloidal non-metallic particles such as selenium, dyed orcolored particles such as a dyed plastic or a stained microorganism,colored or colorable organic polymer latex particles, Duracytes®(derivatized red blood cells, available from Abbott Laboratories, AbbottPark, Ill.), liposomes or other vesicles containing directly visiblesubstances, and the like. The selection of a particular label is notcritical. The label will be capable of producing a signal either byitself (such as a visually detectable colored organix polymer latexparticle) or instrumentally detectable (such as a luminescent compoundor radiolabeled element) or detectable in conjunction with one or moreadditional substances such as an enzyme/substrate signal producingsystem. A variety of different labeled reagents can be formed by varyingeither the label or the specific binding member component of the labeledreagent; it will be appreciated by one skilled in the art that thechoice involves consideration of the analyte to be detected with thedesired means of detection.

When using a visually detectable particle as the label, such asselenium, dyed particles or black latex, the labeled reagent bindingmember(s) may be attached to the particles. Alternatively, the bindingmember(s) may be attached to separate batches of particles andafterwards the particles mixed.

"Signal producing component" refers to any substance capable of reactingwith another assay reagent or with the analyte to produce a reactionproduct or signal that indicates the presence of the analyte and/orserves to indicate that certain assay characteristics have beensatisfied. The signal producing component is detectable by visual orinstrumental means. "Signal production system" as used herein refers tothe group of assay reagents that are needed to produce the desiredreaction product or signal. Thus, one or more signal producingcomponents can be reacted with the label to generate a detectablesignal. For example, when the label is an enzyme, amplification of thedetectable signal is obtained by reacting the enzyme with one or moresubstrates or additional enzymes and substrates to produce a detectablereaction product.

In a preferred embodiment of the present invention, a visuallydetectable label is used as the label component of the labeled reagent,thereby providing for the direct visual or instrumental readout of thepresence or amount of the analyte in the test sample without the needfor additional signal producing components at the detection sites.Suitable materials for use include colloidal metals such as gold and dyeparticles as well as non-metallic colloids such as colloidal selenium,tellurium and sulfur particles.

"Immobilized capture reagent" refers to one or more specific bindingmembers that are attached within or upon a portion of the solid phasesupport or chromatographic strip to form one or more "capture sites"wherein the analyte, positive control reagent, and/or labeled reagentbecome immobilized on the strip or wherein the immobilized reagent slowsthe migration of the analyte and/or labeled reagent through the strip.The method of attachment is not critical to the present invention. Theimmobilized capture reagent facilitates the observation of thedetectable signal by substantially separating the analyte and/or thelabeled reagent from unbound assay reagents and the remaining componentsof the test sample. In addition, the immobilized reagent may beimmobilized on the solid phase before or during the performance of theassay by means of any suitable attachment method.

Typically, a capture site of the present invention is a delimited ordefined portion of the solid phase support such that the specificbinding reaction between the immobilized capture reagent and analyte.This facilitates the detection of label that is immobilized at thecapture site or sites in contrast to other portions of the solid phasesupport. The delimited site is typically less than 50% of the solidphase support, and preferably less than 10% of the solid phase support.The immobilized reagent can be applied to the solid phase material bydipping, inscribing with a pen, dispensing through a capillary tube orthrough the use of reagent jet-printing or biodotting or any othersuitable dispensing techniques. In addition, the capture site can bemarked, for example with a dye, such that the position of the capturesite upon the solid phase material can be visually or instrumentallydetermined even when there is no label immobilized at the site.Preferably, the immobilized reagent is positioned on the strip such thatthe capture site is not directly contacted with the test sample, thatis, the test sample must migrate by capillary action through at least aportion of the strip before contacting the immobilized reagent.

The immobilized capture reagent may be provided in a single capture ordetection site or in multiple sites on or in the solid phase material.The preferred embodiment of the invention provides for immobilizedpatient capture reagent(s) and an immobilized procedural capturereagent. The immobilized capture reagents may also be provided in avariety of configurations to produce different detection or measurementformats. For example, the immobilized capture reagent may be configuredas a letter, number, icon or symbol or any combination thereof. Whenconfigured as a letter, the immobilized capture reagent may be either asingle letter or combination of letters that form words or abbreviatedwords such as "POS", "NEG" or "OK". Alternatively, the immobilizedcapture reagent may be configured as a symbol or combination of symbols,such as for example, a plus, minus, check-mark, bar, diamond, triangle,rectangle, circle, oval, square, arrow, line or any combination thereof.The immobilized capture reagent can be provided as a discreet capturesite or "band" of reagent on or in the solid phase material.Alternatively, the immobilized reagent can be distributed over a largeportion of the solid phase material in a substantially uniform manner toform the capture site. The extent of signal production in the patientcapture site is related to the amount of analyte in the test sample.When using a positive control, the extent of signal production in apositive control capture site, if desired, is related to the amount ofpositive control reagent applied to the strip.

"Negative binding reagent" which may be used interchangeably with theterms "negative control" or "negative control reagent" refers to anysubstance which is used to determine the presence of non-specificbinding or aggregation of any labeled reagent. The negative controlreagent may be, for example, a substance comprising specific bindingmembers such as antigens, antibodies or antibody fragments.Additionally, the negative control reagent may be derived from the sameor a different species as the other reagents on the teststrip or from acombination of two or more species. The presence of a detectable signalfrom the negative control reagent on the teststrip indicates an invalidtest.

"Ancillary specific binding member" refers to any member of a specificbinding pair which is used in the assay in addition to the specificbinding members of the indicator reagent or immobilized capture reagent.One or more ancillary specific binding members can be used in an assay.For example, an ancillary specific binding member can be capable ofbinding the indicator reagent to the analyte of interest, in instanceswhere the analyte itself could not directly attach to the indicatorreagent. Alternatively, an ancillary specific binding member can becapable of binding the immobilized capture reagent to the analyte ofinterest, in instances where the analyte itself could not directlyattach to the immobilized capture reagent. The ancillary specificbinding member can be incorporated into the assay device or it can beadded to the device as a separate reagent solution.

The "solid phase support" or "chromatographic material" or "strip"refers to any suitable porous, absorbent, bibulous, isotropic orcapillary material, which includes the reaction site of the device andthrough which the analyte or test sample can be transported by acapillary or wicking action. It will be appreciated that the strip canbe made of a single material or more than one material (e.g., differentzones, portions, layers, areas or sites can be made of differentmaterials) so long as the multiple materials are in fluid-flow contactwith one another thereby enabling the passage of test sample between thematerials. Fluid-flow contact permits the passage of at least somecomponents of the test sample, e.g., analyte, between the zones of theporous material and is preferably uniform along the contact interfacebetween the different zones.

Thus, natural, synthetic or naturally occurring materials that aresynthetically modified can be used as the solid-phase support andinclude, but are not limited to: papers (fibrous) or membranes(microporous) of cellulose materials such as paper, cellulose, andcellulose derivatives such as cellulose acetate and nitrocellulose;fiberglass; cloth, both naturally occurring (e.g., cotton) and synthetic(e.g., nylon); porous gels; and the like. The porous material should notinterfere with the production of a detectable signal. Thechromatographic material may have an inherent strength, or strength canbe provided by means of a supplemental support.

The particular dimensions of the strip material is a matter ofconvenience, depending upon the size of the test sample involved, theassay protocol, the means for detecting and measuring the signal, andthe like. For example, the dimensions may be chosen to regulate the rateof fluid migration as well as the amount of test sample to be imbibed bythe chromatographic material.

When appropriate, it is necessary to select strip dimensions that allowthe combination of multiple strips in a single assay device. It also iswithin the scope of this invention to have a reagent, at the distal endof the chromatographic material, which indicates the completion of abinding assay (i.e., end of assay indicator) by hanging color uponcontact with the test solution, wicking solution or a signal producingcomponent. Reagents which would change color upon contact with a testsolution containing water are the dehydrated transition metal salts suchas CuSO₄, Co(NO₃)₂, and the like. pH indicator dyes also can be selectedto respond to the pH of the buffered wicking solution. For example,phenolphthalein changes from clear (i.e., colorless) to intense pinkupon contact with a wicking solution having a pH range between 8.0-10.0.

Capture reagents may be located anywhere along the teststrip in singleor multiple pathways with the proviso that they be located in the fluidflow path of their respective labeled reagents. It is understood bythose skilled in the art that as fluid migrates through the strip thereis little cross flow of fluid. Thus, all mobile reagents coming intocontact with the fluid also migrate in the direction of the fluid flow,i.e. there is no substantial migration of reagents transversely acrossthe strip.

The present invention further provides kits for carrying out bindingassays. For example, a kit according to the present invention cancomprise a teststrip such as the teststrip depicted in FIG. 12, oralternatively can comprise the comb-type or card-type device with itsincorporated reagents as well as a transport solution and/or test samplepretreatment reagent as described above. Other assay components known tothose skilled in the art include buffers, stabilizers, detergents,bacteria inhibiting agents and the like which can also be present in theassay device or separate reagent solution.

The present invention optionally includes a non-reactive cover (alsoreferred to as an enclosure or casing) around the device. Preferably,the cover encloses at least the strip to avoid contact with andcontamination of the capture sites. The cover also may include a raisedarea adjacent to the application pad to facilitate receiving and/orcontaining a certain volume of the test sample and/or wicking solution.Additionally, the cover may include a cut out area or areas in the formof a letter, number, icon, or symbol or any combination thereof. In thisembodiment, the cut out area or areas form the design for particularcapture site or sites once the strip is completely enclosed. It ispreferred that a sufficient portion of the strip be encased to preventapplied test sample from contacting the capture sites without firstpassing through a portion of the strip.

Another device component is a test sample application pad, which may bean optional feature. The application pad is in fluid flow contact withone end of the strip material, referred to as the proximal end, suchthat the test sample can pass or migrate from the application pad to thestrip. Fluid flow contact can include physical contact of theapplication pad to the chromatographic material, as well as theseparation of the pad from the strip by an intervening space oradditional material which still allows fluid to pass between the pad andthe strip. Substantially all of the application pad can overlap thechromatographic material to enable the test sample to pass throughsubstantially any part of the application pad to the proximal end of thestrip. Alternatively, only a portion of the application pad might be influid flow contact with the chromatographic material. The applicationpad can be any material which can transfer the test sample to thechromatographic material and which can absorb a volume of test samplethat is equal to or greater than the total volume capacity of thechromatographic material.

Materials preferred for use in the application pad includenitrocellulose, porous polyethylene frit or pads and glass fiber filterpaper. The material also must be chosen for its compatibility with theanalyte and assay reagents.

In addition, the application pad typically contains one or more assayreagents either diffusively or non-diffusively attached thereto.Reagents which can be contained in the application pad include, but arenot limited to, labeled reagents, ancillary specific binding members,and signal producing system components needed to produce a detectablesignal. For example, in a binding assay it is preferred that the labeledreagent be contained in the application pad. The labeled reagent isreleased from the pad to the strip with the application of the testsample, thereby eliminating the need to combine the test sample andlabeled reagent prior to using the device. The isolation of assayreagents in the application pad also keeps separate the interactivereagents and facilitates the manufacturing process.

In some instances, the application pad also serves the function of aninitial mixing site and a reaction site for the test sample and reagent.In preferred embodiments, the application pad material is selected toabsorb the test sample at a rate that is faster than that achieved bythe strip material alone. Typically, the pad material is selected toabsorb fluids two to five times faster than the strip material.Preferably, the pad will absorb fluids four to five times faster thanwill the strip material.

In an optional embodiment of the present invention, gelatin is used toencompass all or part of the application pad. Typically, suchencapsulation is produced by overcoating the application pad with fishgelatin. The effect of this overcoating is to increase the stability ofthe reagent contained by the application pad. The application of testsample to the overcoated application pad causes the gelatin to dissolveand thereby enables the dissolution of the reagent. In anotherembodiment of the present invention, the reagent containing applicationpad is dried or lyophilized to increase the shelf-life of the device.Lyophilized application pads have been found to produce stronger signalsthan air-dried application pads, and the lyophilized application padshave been found to maintain stability for longer periods of time. Thereagents contained in the application pad are rehydrated with theaddition of test sample to the pad.

The present invention also can be modified by the addition of afiltration means. The filtration means can be a separate material placedabove the application pad or between the application pad and the stripmaterial, or the material of the application pad itself can be chosenfor its filtration capabilities. The filtration means can include anyfilter or trapping device used to remove particles above a certain sizefrom the test sample. For example, the filter means can be used toremove red blood cells from a sample of whole blood, such that plasma isthe fluid received by the application pad and transferred to thechromatographic material.

Yet another modification of the present invention involves the use of anadditional layer or layers of porous material placed between theapplication pad and the chromatographic material or overlaying theapplication pad. Such an additional pad or layer can serve as a means tocontrol the rate of flow of the test sample from the application pad tothe strip. Such flow regulation is preferred when an extended incubationperiod is desired for the reaction of the test sample and the reagent(s)in the application pad. Alternatively, such a layer can containadditional assay reagent(s) that preferably is isolated from theapplication pad reagent(s) until the test sample is added, or it canserve to prevent unreacted assay reagents from passing to thechromatographic material.

When small quantities of non-aqueous or viscous test samples are appliedto the application pad, it may be necessary to employ a wicking ortransport solution, preferably a buffered solution, to carry thereagent(s) and test sample from the application pad and through thestrip. When an aqueous test sample is used, a transport solutiongenerally is not necessary but can be used to improve flowcharacteristics through the device or to adjust the pH of the testsample. The transport solution typically has a pH range from about 5.5to about 10.5, and more preferably from about 6.5 to about 9.5. The pHis selected to maintain a significant level of binding affinity betweenthe specific binding members in a binding assay. When the labelcomponent of the indicator reagent is an enzyme, however, the pH alsomust be selected to maintain significant enzyme activity for colordevelopment in enzymatic signal production systems. Illustrative buffersinclude phosphate, carbonate, barbital, diethylamine,tris(hydromethyl)aminomethane (Tris), Bis-Tris,2-amino-2-methyl-1-propanol and the like. The transport solution and thetest sample can be combined prior to contacting the application pad orthey can be contacted to the application pad sequentially.

Predetermined amounts of signal producing components and ancillaryreagents can be incorporated within the device, thereby avoiding theneed for additional protocol steps or reagent additions. Thus, it alsois within the scope of this invention to provide more than one reagentto be immobilized within the application pad and/or the strip material.

This invention provides assay devices and methods, where the devices usestrips of chromatographic material capable of transporting liquids forthe performance of an assay on a patient sample or the performance of amultiple assay on a patient sample. The device may include test sampleapplication pads in fluid flow contact with the strip which function toregulate the flow of test sample to the chromatographic material, tofilter the test samples and to deliver and/or mix assay reagents. Assayreagents may be incorporated within the application pad as well as inthe chromatographic material. By varying the configuration ofreagent-containing sites on the device, qualitative and quantitativedisplays of assay results can be obtained. Preferably, the reagents aresituated in the devices in such a way as to make the assay substantiallyself-performing and to facilitate the detection and quantitation of theassay results. One or more detectable signals resulting from thereactions at the reagent-containing sites and/or the binding assay thencan be detected by instrumentation or direct visual observation.

The present invention provides an assay for simultaneously detecting anddifferentiating antibodies to HIV-1 group M, HIV-1 group O and HIV-2 ina test sample, and an analytical device with which to perform thissimultaneous detection and differentiation. In a sandwich assay format,the test sample suspected of containing the analyte (for example,antibody to HIV-1 group M) is contacted with a predetermined amount ofindicator reagent (in this example, labeled anti-species antibody Ab*!)to form a reaction mixture containing an analyte/indicator reagentcomplex (Ab-Ab*). The indicator reagent (Ab*) may be separate from orpreferably incorporated within the test device. The resulting reactionmixture then migrates through the teststrip. The reaction mixturecontacts capture reagent sites (one for HIV-1 group M, one for HIV-1group O, and one for HIV-2) containing separately immobilized analytespecific binding member ( l-Ag!) that binds at a site on the analytedistinct from the indicator reagent. The capture reagent therefore iscapable of binding to the Ab-Ab* complex to form an immobilizedl-Ab-Ag-Ab* complex that is detectable at the capture reagent site.Furthermore, the reaction mixture also may migrate further through theteststrip and react with reagent present in the end of assay indicatorsite.

Referring to FIG. 13, the test device (18) for the assay comprises anitrocellulose membrane strip (24) upon which are placed and allowed todry in separate distinct capture areas, selected specific and highlypurified recombinant antigens derived from the HIV-1 group M (26), HIV-1group O (28) and HIV-2 gp41 (30) region of each. The test device (18)further comprises a conjugate pad (32) which comprises a glass fiberfilter (34) presenting a selenium colloid sensitized with ananti-species antibody (e.g., goat anti-human IgG) suspended in a fluidcontaining nitrocellulose blocking proteins which has been dried beforeassembly and affixed to the distal end (20) of the nitrocellulosemembrane (24). The entire device (18) is held permanently in place by atop clear laminating material (36) which bears an adhesive surface (38)in contact with the top surface of the nitrocellulose membrane (24) andattached to the conjugate pad (20), and a bottom laminating material(48) which bears an adhesive surface (38) in contact with the bottomsurface (48) of the nitrocellulose membrane (24). The test fluid flowsfrom the distal end (20) to the proximal end (22) and contacts each ofthe three separate distinct capture areas. The device also can have atest sample pad and reactivity zone (40) upon which anti-species (i.e.,anti-human) conjugate is placed. The device also preferably has ablotter (44) to absorb any remaining fluid in the device and has a sitefor indicating completion of the assay (46). The read out (in thecapture areas and/or in the test sample reactivity zone) can be eithervisual direct readout without the aid of laboratory equipment orautomated by an instrument. Furthermore, the test device can be enclosedin a casing (42) of molded plastic or other suitable material.

The assay is performed as follows. Test sample such as human serum,preferably previously diluted in buffer (elution buffer, consisting of50 mM TRIS (pH 8.4), 1% w/v solid bovine serum albumin BSA!, 0.4% v/vTriton X-405®, 1.5% w/v casein, 3% w/v bovine IgG, 4% w/v E. colilysate, pH 8.2; dilution at 1 μl serum to 100 μl of elution buffer), iscontacted with the anti-IgG colloid conjugate at the distal end (20) ofthe test device. IgG in the test sample is bound by the anti-IgGcolloid, and the complexes are chromatographed along the length of theabsorbant pad (preferably, nitrocellulose membrane). As the complexesflow, they pass over the discrete zones (FIG. 13, sites 30, 26, and 28)in which the HIV recombinant antigens previously have been applied. Ifthe complexes contain specific antibody to the recombinant antigens inany of the discrete zones, a reaction takes place and red zones of colorappear in the appropriate zone(s). Multiple specificities can bedetermined simultaneously. In addition, a positive control, consistingof a pooled test sample positive for all three antigens tested, shouldreact positively in all three zones. Alternatively, a positive controlsample, reactive with each of the antigens in the test, can be runseparately for each analyte for which antibody is being assayed.

It is contemplated and within the scope of the present invention thatantibody analytes to HIV-1 group M, HIV-1 group O, and HIV-2, may bedetectable in these assays by use of a synthetic, recombinant orpurified polypeptide comprising the entire or partial polypeptide (aminoacid) sequences described herein, as the capture reagent. "Purifiedprotein" (or "purified polypeptide") means a polypeptide of interest orfragment thereof which is essentially free, that is, contains less thanabout 50%, preferably less than about 70%, and more preferably, lessthan about 90%, of cellular components with which the polypeptide ofinterest is naturally associated. Methods for purifying are known in theart. A "recombinant polypeptide" or "recombinant protein" or"polypeptide produced by recombinant techniques," which are usedinterchangeably herein, describes a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system. Further, the term "synthetic peptide" asused herein means a polymeric form of amino acids of any length, whichmay be chemically synthesized by methods well-known to the routineer.These synthetic peptides are useful in various applications.

The preferred capture reagent for HIV-1 group O comprises a polypeptidesequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO: 52, and SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60,the capture reagent for HIV-1 group M comprises SEQ ID NO: 56, and thecapture reagent for HIV-2 comprises SEQ ID NO: 55. It is preferred thatthese polypeptides be produced by recombinant technology.

The present invention will now be described by way of examples, whichare meant to illustrate, but not to limit, the spirit and scope of theinvention.

EXAMPLES Example 1 Cloning

Oligonucleotides for gene construction and sequencing were synthesizedat Abbott Laboratories, Synthetic Genetics (San Diego, Calif.) or OligoEtc. (Wilsonville, Calif.). All polymerase chain reaction (PCR)reagents, including AmpliTaq DNA polymerase and UlTma DNA polymerase,were purchased from Perkin-Elmer Corporation (Foster City, Calif.) andused according to the manufacturer's specifications unless otherwiseindicated. PCR amplifications were performed on a GeneAmp 9600 thermalcycler (Perkin-Elmer). Unless indicated otherwise, restriction enzymeswere purchased from New England BioLabs (Beverly, Mass.) and digestswere performed as recommended by the manufacturer. DNA fragments usedfor cloning were isolated on agarose (Life Technologies, Gaithersburg,Md.) gels, unless otherwise indicated.

Desired fragments were excised and the DNA was extracted with a QIAEX IIgel extraction kit or the QIAquick gel extraction kit (Qiagen Inc.,Chatsworth, Calif.) as recommended by the manufacturer. DNA wasresuspended in H₂ O or TE 1 mM ethylenediaminetetraacetic acid (EDTA; pH8.0; BRL Life Technologies), 10 mMtris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCI; pH 8.0; BRLLife Technologies)!. Ligations were performed using a Stratagene DNAligation kit (Stratagene Cloning Systems, La Jolla, Calif.) asrecommended by the manufacturer. Ligations were incubated at 16° C.overnight.

Bacterial transformations were performed using MAX EFFICIENCY DH5αcompetent cells (BRL Life Technologies) or Epicurian Coli XL1-Bluesupercompetent cells (Stratagene Cloning Systems) following themanufacturer's protocols. Unless indicated otherwise, transformationsand bacterial restreaks were plated on LB agar (Lennox) plates with 150μg/ml ampicillin (M1090; MicroDiagnostics, Lombard, Ill.) or on LBagar+ampicillin plates supplemented with glucose to a finalconcentration of 20 mM, as noted. All bacterial incubations (plates andovernight cultures) were conducted overnight (˜16 hours) at 37° C.

Screening of transformants to identify desired clones was accomplishedby sequencing of miniprep DNA and/or by colony PCR. Miniprep DNA wasprepared with a Qiagen Tip 20 Plasmid Prep Kit or a Qiagen QIAwell 8Plasmid Prep Kit following the manufacturer's specifications, unlessotherwise indicated. For colony PCR screening, individual colonies werepicked from transformation plates and transferred into a well in asterile flat-bottom 96-well plate (Costar, Cambridge, Mass.) containing100 μl sterile H₂ O. One-third of the volume was transferred to a secondplate and stored at 4° C. The original 96-well plate was microwaved for5 minutes to disrupt the cells. 1 μl volume then was transferred to aPCR tube as template. 9 μl of a PCR master mix containing 1 μl 10X PCRbuffer, 1 μl 2 mM dNTPs, 1 μl (10 pmol) sense primer, 1 μl (10 pmol)anti-sense primer, 0.08 μl AmpliTaq DNA polymerase (0.4 units), and 4.2μl H₂ O was added to the PCR tube. Reactions were generally amplifiedfor 20-25 cycles of 94° C. for 30 seconds, 50-60° C. (depending onprimer annealing temperatures) for 30 seconds and 72° C. for 60 seconds.Primers were dependent on the insert and cycle conditions were modifiedbased on primer annealing temperatures and the length of the expectedproduct. After cycling, approximately 1/3 of the reaction volume wasloaded on an agarose gel for analysis. Colonies containing desiredclones were propagated from the transfer plate.

Unless otherwise indicated, DNA sequencing was performed on an automatedABI Model 373 Stretch Sequencer (Perkin Elmer). Sequencing reactionswere set up with reagents from a FS TACS Dye Term Ready Reaction Kit(Perkin Elmer) and 250-500 ng plasmid DNA according to themanufacturer's specifications. Reactions were processed on Centri-Sepcolumns (Princeton Separations, Adelphia, N.J.) prior to loading on theSequencer. Sequence data was analyzed using Sequencher 3.0 (Gene CodesCorporation, Ann Arbor, Mich.) and GeneWorks 2.45 (Oxford MolecularGroup, Inc., Campbell, Calif.).

Example 2 Determination of the Env Sequence of the HIV-1 Group O IsolateHAM112.

Viral RNA was extracted from culture supernatants of human peripheralblood mononuclear cells infected with the HIV-1 group O isolatedesignated HAM 112 (H. Hampl et al., supra) using a QIAamp Blood Kit(Qiagen) and the manufacturer's recommended procedure. RNA was eluted ina 50 μl volume of nuclease-free water (5Prime-3Prime, Inc., Boulder,Colo.) and stored at -70° C. The strategy for obtaining the env regionsequence involved cDNA synthesis and PCR (nested) amplification of fouroverlapping env gene fragments. The amplified products were sequenceddirectly on an automated ABI Model 373 Stretch Sequencer. Amplificationreactions were carried out with GeneAmp RNA PCR and GeneAmp PCR Kits(Perkin Elmer) as outlined by the manufacturer. Oligonucleotide primerpositions correspond to the HIV-1 ANT70 env sequence (G. Myers et al.,eds., supra). The primers env10R nucleotide (nt) 791-772; SEQ ID NO:62!, env15R (nt 1592-1574; SEQ ID NO: 63), env22R (nt 2321-2302; SEQ IDNO: 64), env26R (nt 250-232 3' of env; SEQ ID NO: 65) were used for cDNAsynthesis of fragments 1-4, respectively. Reverse transcriptionreactions were incubated at 42° C. for 30 minutes then at 99° C. for 5minutes. First round PCR amplifications consisted of 30 cycles of 95° C.for 30 seconds, 52° C. for 30 seconds, and 72° C. for 1 minute using theprimer combinations: env1F (nt 184-166 5' of env; SEQ ID NO: 66) andenv10R (SEQ ID NO: 62), env7F (nt 564-586; SEQ ID NO: 67) and env15R(SEQ ID NO: 63), env12F (nt 1289-1308; SEQ ID NO: 68) and env22R (SEQ IDNO: 64), env19F (nt 2020-2040; SEQ ID NO: 69) and env26R (SEQ ID NO: 65)for fragments 1 through 4, respectively. For the second round ofamplification (nested PCR), 5 μl of the respective first round PCRreactions was used as template along with the primer combinations env2F(nt 37-15 5' of env; SEQ ID NO: 70) and env9R (nt 740-721; SEQ ID NO:71), env8F (nt 631-650; SEQ ID NO: 72) and env14R (nt 1437-1416; SEQ IDNO: 73), env13F (nt 1333-1354; SEQ ID NO: 74) and env21R (nt 2282-2265;SEQ ID NO: 75), env20F (nt 2122-2141; SEQ ID NO: 76) and env25R (nt111-94 3' of env; SEQ ID NO: 77) for fragments 1 through 4,respectively. Second round amplification conditions were identical tothose used for the first round. Fragments were agarose gel-purified andextracted with a Qiagen QIAEX II Gel Extraction Kit. Fragments weresequenced directly with the primers used for nested PCR along withprimers env4F (SEQ ID NO: 78) and env5R (SEQ ID NO: 79) for fragment 1;primers env10F (SEQ ID NO: 80), env12F (SEQ ID NO: 81), env11R (SEQ IDNO: 82), env12F (SEQ ID NO: 68), and AG1 (SEQ ID NO: 87) for fragment 2;primers env15F (SEQ ID NO: 83) and env19R (SEQ ID NO: 84) for fragment3; primers env22F (SEQ ID NO: 85) and env24R (SEQ ID NO: 86) forfragment 4. The deduced amino acid sequence of env from the HIV-2 groupO isolate HAM112 (SEQ ID NO: 61) is presented in FIG. 1.

Example 3 Construction of Synthetic HIV-1 Group O env gp120/gp41 Genes

FIG. 2 depicts the strategy used to generate synthetic HIV-1 group O envgp120/gp41 gene constructs. The env gp120/gp41 sequences were based onthe HIV-1 group O isolate HAM112 (SEQ ID NO: 61) (H. Hampl et al.).Determination of the env sequence of HAM112 is outlined in Example 2,hereinabove. Oligonucleotides were designed that encode the C-terminal45 amino acids of the env gp120 and 327 amino acids of env gp41(nucleotide #1 is the first base of the first codon of gp120 in thesynthetic gene). The synthetic gene has a 26 amino acid deletion(nucleotides 643 through 720), relative to the native HAM112 gp41, thatencompasses a highly hydrophobic (H) region (transmembrane region) ofgp41. Thus, the full-length synthetic gp41 gene constructed is 327 aminoacids.

In the synthetic oligonucleotides, the native HIV-1 codons were alteredto conform to E. coli codon bias in an effort to increase expressionlevels of the recombinant protein in E. coli. See, for example, M. Gouyand C. Gautier, Nucleic Acids Research 10:7055 (1982); H. Grosjean andW. Fiers, Gene 18:199 (1982); J. Watson et al. (eds.), Molecular Biologyof the Gene, 4th Ed., Benjamin Kumming Publishing Co., pp.440 (1987).The gene construction strategy involved synthesis of a series ofoverlapping oligonucleotides with complementary ends (Osyn-A throughOsyn-L, depicted as A through L). When annealed, the ends served asprimers for the extension of the complementary strand.

The fragments then were amplified by PCR. This process ("PCR knitting"of oligonucleotides) was reiterated to progressively enlarge the genefragment. Oligonucleotide Osyn-5' was designed for cloning into the pLvector pKRR826. The expression vector, pKRR826, is a modified form ofthe lambda pL promoter vector pSDKR816, described in U.S. Ser. No.08/314,570, incorporated herein by reference. pKRR826 is a high copynumber derivative of pBR322 that contains the temperature sensitive cIrepressor gene (Benard et al., Gene 5:59 1979!). However, pKRR826 lacksthe translational terminator rrnBt1 and has the lambda pL and lambda pRpromoters in the reverse orientation, relative to pSDKR816. Thepolylinker region of pKRR826 contains Eco RI and Bam HI restrictionenzyme sites and lacks an ATG start codon. Optimal expression isobtained when the 5' end of the gene insert (including an N-terminalmethionine) is cloned into the EcoRI site.

Osyn-5' was designed to contain an Eco RI restriction site for cloningand an ATG codon (methionine) to provide for proper translationalinitiation of the recombinant proteins. The anti-sense oligonucleotidesOsyn-O3'(SEQ ID NO: 15), Osyn-P3' (SEQ ID NO: 16), and Osyn-M (M) (SEQID NO: 14) each contain two sequential translational termination codons(TAA,TAG) and a Bam HI restriction site. When outside primers Osyn-5'(SEQ ID NO: 11) and Osyn-M (M) (SEQ ID NO: 14) were used, a full-lengthgp41 (327 amino acids) gene was synthesized (pGO-11PL; SEQ ID NO: 52).Outside oligonucleotides Osyn-5' (SEQ ID NO: 11) and OsynO3' (SEQ ID NO:15) resulted in a truncated gp41 product of 199 amino acids (pGO-9PL;SEQ ID NO: 48). Alternatively, outside oligonucleotides Osyn-5' (SEQ IDNO: 11) and Osyn-P3' (SEQ ID NO: 16) resulted in a truncated gp41product 169 amino acids in length (pGO-8PL; SEQ ID NO: 58).

The synthetic genes also were expressed as CMP-KDO synthetase (CKS)fusion proteins. PCR-mediated transfer of the synthetic genes frompKRR826 into pJO200 (described in U.S. Ser. No. 572,822, andincorporated herein by reference) was accomplished with an alternativeoutside sense oligonucleotide PCR primer (5' end), Osyn-5'CKS (SEQ IDNO: 25). Osyn-5'CKS contained an Eco RI restriction site and resulted inthe in-frame fusion of the synthetic gene insert to CKS in theexpression vector pJO200. The 3' outside primers (antisense) Osyn-M (SEQID NO: 14), Osyn-)3' (SEQ ID NO: 15) and Osyn-P3' (SEQ ID NO: 16) wereused in combination with Osyn-5'CKS (SEQ ID NO: 25) to generatepGO-11CKS (SEQ ID NO: 54), pGO-9CKS (SEQ ID NO: 50), and pGO-8CKS (SEQID NO: 60), respectively. These steps are detailed hereinbelow.

A. PCR Knitting of Synthetic Oligonucleotides

Three PCR reactions (100 μl volume) were set up as follows:

(1) Reaction 1B: AmpliTaq DNA polymerase (2.5U) and 1X buffer, alongwith 40 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 25 pmol each ofoligonucleotides Osyn-A (SEQ ID NO: 3) and Osyn-D (SEQ ID NO: 5), and0.25 pmol each of oligonucleotides Osyn-B (SEQ ID NO: 17) and Osyn-C(SEQ ID NO: 4);

(2) Reaction 2A: UlTma DNA Polymerase (3U) and 1X buffer along with 1.5mM MgCl₂, 40 μM of each dNTP, 25 pmol each of oligonucleotides Osyn-E(SEQ ID NO: 6) and Osyn-H (SEQ ID NO: 9), and 0.25 pmol each ofoligonucleotides Osyn-F (SEQ ID NO: 7) and Osyn-G (SEQ ID NO: 8); and

(3) Reaction 3B: UlTma DNA Polymerase (3U) and 1X buffer along with 1.5mM MgCl₂, 40 μM of each dNTP, 25 pmol each of oligonucleotides Osyn-I(SEQ ID NO: 10) and Osyn-L (SEQ ID NO: 13), and 0.25 pmol each ofoligonucleotides Osyn-J (SEQ ID NO: 18) and Osyn-K (SEQ ID NO: 12).

Amplifications consisted of 20 cycles of 97° C. for 30 seconds, 52° C.for 30 seconds and 72° C. for 60 seconds. Reactions were then incubatedat 72° C. for 7 minutes and held at 4° C. PCR-derived products 1B, 2Aand 3B were gel isolated on a 1% agarose gel.

B. PCR Knitting of PCR Products From Reaction 1B and Reaction 2A.

A PCR reaction was set up with UlTma DNA Polymerase (3U) and 1X bufferalong with 1.5 mM MgCl₂, 40 μM of each dNTP, 24.4 pmol ofoligonucleotide Osyn-5' (SEQ ID NO: 11), 25 pmol of oligonucleotideOsyn-P3' (SEQ ID NO: 16), and ˜10 ng each of gel-isolated 1B and 2Aproducts from Example 3, Section 1A, hereinabove. Cycling conditionswere the same as in Example 3, Section 1A. A second round ofamplification was used to generate more of the desired product. This wasperformed by making an UlTma mix as described hereinabove (100 μlreaction volume) with 49 pmol Osyn-5' (SEQ ID NO: 11), 50 pmol Osyn-P3'(SEQ ID NO: 16) and 5 μl of the PCR product from the first round astemplate. These reactions were incubated at 94° C. for 90 seconds, andthen used cycled as above (Section 3A). The Osyn-5'/Osyn-P3' PCR productwas gel-isolated as described hereinabove.

C. Cloning of the Osyn-5'-Osyn-P3' PCR Product.

The Osyn-5'-Osyn-P3' PCR product was digested with the restrictionendonucleases Eco RI+Bam HI and ligated into the vector pKRR826(described hereinabove) that had been digested with Eco RI+Bam HI andgel-isolated. The ligation product was used to transform DH5α competentcells. The desired clone was identified by colony PCR usingoligonucleotides pKRREcoRI Forward (SEQ ID NO: 38) and pKRRBamHI Reverse(SEQ ID NO: 39). Miniprep DNA was prepared from an overnight culture ofpGO-8 candidate clone A2 and the Osyn-5'Osyn-P3' plasmid insert wassequenced with the oligonucleotide primers pKRREcoRI Forward (SEQ ID NO:38), pKRRBamHI Reverse (SEQ ID NO: 39), 41sy-1(SEQ ID NO: 44), and41sy-2(SEQ ID NO: 41).

D. Modification of pGO-8 Candidate Clone A2.

A 100 μl volume PCR reaction was set up with UlTma DNA Polymerase (3U)and 1X buffer, along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 pmol ofoligonucleotides Osyn-5'-repair (SEQ ID NO: 24), 50 pmol Osyn-P3' (SEQID NO: 16), and ˜1 ng of pGO-8 candidate clone miniprep DNA as templateA2 (obtained from the reactions set forth hereinabove). The reaction wasincubated at 94° C. for 90 seconds, and then amplified with 20 cycles of94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 60 seconds.The Osyn-5'-repair/Osyn-P3'PCR product then was gel isolated anddigested with Eco RI+Bam HI. The digested product was ligated into EcoRI+Bam HI digested pKRR826 vector. The ligation product was used totransform DH5α competent cells. The desired clone was identified bycolony PCR using oligonucleotides pKRREcoRI Forward (SEQ ID NO: 38) andpKRRBamHI Reverse (SEQ ID NO: 39). An overnight culture of pGO-8candidate clone 6 was set up and a miniprep DNA was prepared. The Osyn5'repair/Osyn-P3' plasmid insert was sequenced with the oligonucleotideprimers pKRREcoRI Forward (SEQ ID NO: 38), pKRRBamHI Reverse (SEQ ID NO:39), 41sy-1(SEQ ID NO: 44), and 41sy-2(SEQ ID NO: 41). Based on thesequencing results, pGO-8 candidate clone #6 was designatedpGO-8PL/DH5α. SEQ ID NO: 57 presents the nucleotide sequence of thecoding region. FIG. 5 presents the amino acid sequence of the pGO-8PLrecombinant protein (SEQ ID NO: 58). The pGO-8PL recombinant proteinconsists of a N-terminal methionine, 45 amino acids of env gp120 (HIV-1group O, HAM112 isolate), and 169 amino acids of env gp41 (HIV-1 groupO, HAM112 isolate).

E. Construction of pGO-8CKS/XL1.

pGO-8CKS/XL1 (SEQ ID NO: 59 presents the nucleotide sequence of thecoding region) encodes the recombinant protein pGO-8CKS. FIG. 6 presentsthe amino acid sequence of pGO-8CKS (SEQ ID NO: 60). This proteinconsists of 246 amino acids of CKS/polylinker, 45 amino acids of envgp120 (HIV-1 group O, HAM112 isolate), and 169 amino acids of env gp41(HIV-1 group O, HAM112 isolate). The construction of pGO-8CKS/XL1 wasaccomplished as follows.

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U)and 1X buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 pmol ofOsyn-5'CKS (SEQ ID NO: 25), 50 pmol Osyn-P3' (SEQ ID NO: 16), and 1 ngpGO-8PL clone #6 miniprep DNA. The reaction was incubated at 94° C. for90 seconds then amplified with 25 cycles of 94° C. for 30 seconds; 55°C. for 30 seconds; 72° C. for 90 seconds. Then, the Osyn-5'CKS/Osyn-P3'PCR product was gel isolated. EcoR I+Bam HI digested theOsyn-5'CKS/Osyn-P3' PCR product and the vector pJO200. The digestedpJO200 vector was gel isolated and ligated to digestedOsyn-5'CKS/Osyn-P3' PCR product. XL1-Blue supercompetent cells weretransformed with the ligation and plated on LB+ampicillin platessupplemented with 20 mM glucose. Colonies were restreaked for isolationon the same type of plates. An overnight culture of clone pGO-8CKS/XL1was grown in LB broth+100 μg/ml carbenicillin (Sigma Chemical Co.)+20 mMglucose (Sigma Chemical Co.). Frozen stocks (0.5 ml overnightculture+0.5 ml glycerol) were made and DNA was prepared for sequenceanalysis. The following oligonucleotides were used as sequencingprimers: CKS-1(SEQ ID NO: 30), CKS-2(SEQ ID NO: 31), CKS-3(SEQ ID NO:32), CKS-4(SEQ ID NO: 33), 43461 (SEQ ID NO: 2), 43285 (SEQ ID NO: 1),41sy-1B (SEQ ID NO: 29), 41sy-2B (SEQ ID NO: 34), CKS176.1 (SEQ ID NO:19), and CKS3583 (SEQ ID NO: 20).

F. Construction of pGO-9PL/DH5α.

FIGS. 3A through 3D and show a diagrammatic representation of the stepsinvolved in construction of pGO-9PL/DH5α. pGO-9PL/ DH5α encodes therecombinant protein pGO-9PL. SEQ ID NO: 47 present the nucleotidesequence of the coding region of pGO-9PL/DH5α. FIG. 7 illustrates theamino acid sequence of the pGO-9PL recombinant protein (SEQ ID NO: 48).This protein consists of an N-terminal methionine, 45 amino acids of envgp120 (HIV-1 group O, HAM112 isolate), and 199 amino acids of env gp41(HIV-1 group O, HAM112 isolate). Construction of pGO-9PL/DH5α wasaccomplished as follows.

Step 1. A 100 μl PCR reaction was set up with UlTma DNA Polymerase (3U)and 1X buffer, along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 pmol ofOsyn-5' (SEQ ID NO: 11), 50 pmol of Osyn-H (SEQ ID NO: 9), and ˜2 ng ofpGO-8 candidate clone 6 miniprep DNA (obtained from Example 3, Section Dhereinabove) as template. The reaction was incubated at 94° C. for 120seconds, and then amplified with 8 cycles of 94° C. for 30 seconds, 55°C. for 30 seconds and 72° C. for 60 seconds.

Step 2. A 100 μl PCR reaction was set up with UlTma DNA Polymerase (3U)and 1X buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 pmol ofOsyn-5' (SEQ ID NO: 11), 50 pmol Osyn-)3' (SEQ ID NO: 15), and 10 μl ofthe PCR reaction from step 1 as template. The reaction was incubated at94° C. for 120 seconds then amplified with 18 cycles of 94° C. for 30seconds, 55° C. for 30 seconds, 72° C. for 60 seconds, followed byincubation at 72° C. for 5 minutes.

The Osyn-5'/Osyn-O3' PCR product then was gel-isolated and digested withEco RI+Bam HI. The digested product was ligated into Eco RI+Bam HIdigested pKRR826 vector. The ligation product next was used to transformDH5α competent cells. An overnight culture of pGO-9PL candidate clone 3was set up and a miniprep DNA was prepared. The Osyn-5'/Osyn-O3' plasmidinsert was sequenced with the following oligonucleotides as primers:pKRREcoR1 Forward (SEQ ID NO: 38), pKRRBamHI Reverse (SEQ ID NO: 39),41sy-1C (SEQ ID NO: 40), 41sy-2 (SEQ ID NO: 41), 41sy-3 (SEQ ID NO: 42)and 41sy-4 (SEQ ID NO: 23). pGO-9PL clone #3 then was restreaked forisolation. An isolated colony was picked, an overnight culture of it wasgrown, and a frozen stock (0.5 ml glycerol+0.5 ml overnight culture) wasmade. The stock was stored at -80° C. The sequence was confirmed usingthe primers indicated hereinabove, and this clone was designated aspGO-9PL/DH5α (SEQ ID NO: 47 presents the nucleotide sequence of thecoding region, and SEQ ID NO: 48 presents the amino acid sequence ofcoding region). pGO-9PL/DH5α was restreaked, an overnight culture wasgrown, and a miniprep DNA was prepared (this prep was designated as H5).

G. Construction of pGO-9CKS/XL1.

FIG. 3A through 3D show a diagrammatic representation of the stepsinvolved in construction of pGO-9CKS/XL1. pGO-9CKS/XL1 encodes therecombinant protein pGO-9CKS. FIG. 8 presents the amino sequence of thepGO-9CKS recombinant protein (SEQ ID NO: 50). This protein consists of246 amino acids of CKS and polylinker followed by 45 amino acids of envgp120 (HIV-1 group O, HAM112 isolate), and 199 amino acids of env gp41(HIV-1 group O, HAM112 isolate). The construction of pGO-9CKS/XL1 wasaccomplished as follows.

Two PCR reactions (100 μl volume) were set up with UlTma DNA Polymerase(3U) and 1X buffer, along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 pmolof Osyn-5'CKS (SEQ ID NO: 25), 50 pmol Osyn-O3' (SEQ ID NO: 15) and 1 ngpGO-9PL candidate clone 3 miniprep DNA (obtained from Example 3, SectionF, hereinabove). Each reaction was incubated at 94° C. for 120 seconds,then amplified with 24 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 120 seconds, followed by incubation at 72° C. for 5minutes. The Osyn-5'CKS/OsynO3' PCR product then was gel isolated. TheOsyn-5'CKS/Osyn-O3' PCR product and the vector pJO200 was digested withEco RI+Bam HI. The digested pJO200 vector was gel isolated and ligatedto the digested Osyn-5'CKS/Osyn-O3' PCR product. XL1-Blue supercompetentcells were transformed with the ligation and plated on LB+ampicillinplates supplemented with 20 mM glucose. Colonies were restreaked forisolation on the same type of plates. An overnight culture of clonepGO-9CKS candidate clone 4 was grown in LB broth+100 mg/ml carbenicillin(Sigma Chemical Co.)+20 mM glucose (Sigma Chemical Co.). Made frozenstocks (0.5 ml overnight culture+0.5 ml glycerol) and prepared DNA forsequence analysis. The following oligonucleotides were used assequencing primers: CKS-1 (SEQ ID NO: 30), CKS-2 (SEQ ID NO: 31), CKS-3(SEQ ID NO: 32), CKS-4 (SEQ ID NO: 33), 43461 (SEQ ID NO: 2), 43285 (SEQID NO: 1), 41sy-1B (SEQ ID NO: 29), 41sy-2B (SEQ ID NO: 34), 41sy-3B(SEQ ID NO: 35), CKS 176.1 (SEQ ID NO: 19), CKS3583 (SEQ ID NO: 20), andpTB-S8 (SEQ ID NO: 28). Clone pGO-9CKS candidate clone 4 was designatedas pGO-9CKS/XL1 (SEQ ID NO: 49 presents the nucleotide sequence ofcoding region, and SEQ ID NO: 50 presents the amino acid sequence ofcoding region).

H. Construction of Osyn I--M Fragment.

The Osyn--O--M fragment was constructed as follows. A 100 μl PCRreaction was set up using AmpliTaq DNA Polymerase (2.5U), 1X buffer, 50μM of each dNTP, 50 pmol I-PCR (SEQ ID NO: 26), 50 pmol Osyn-M (SEQ IDNO: 14) and 10 ng of gel-isolated PCR fragment 3A (Example 3, section A,hereinabove). The reaction was incubated at 95° C. for 105 seconds, andthen it was amplified with 15 cycles of 95° C. for 30 seconds, 55° C.for 30 seconds, 72° C. for 60 seconds, and then it was held at 72° C.for 7 minutes. The product, designated as Osyn I--M, was gel-isolatedand cloned into the PCR II vector (TA Cloning Kit; Invitrogen, SanDiego, Calif.) following the manufacturer's recommended procedure. Theresulting ligation product was used to transform DH5α competent cells.Plasmid miniprep DNA was generated from an overnight culture of cloneIM-6, and the gene insert was sequenced with oligonucleotides 56759 (SEQID NO: 45) and 55848 (SEQ ID NO: 46).

I. Synthesis and Knitting of PCR Fragments I/6R and IM-6F.

These procedures were performed as follows.

Step 1. The following PCR reactions (100 μl volume) were set up: (a)I/6R with AmpliTaq DNA Polymerase (2.5U), 1X buffer, 50 μM of each dNTP,50 pmol I-PCR (SEQ ID NO: 26), 50 pmol IM-6R (SEQ ID NO: 22) and 281 ngof clone IM-6 (obtained from Example 3, Section H) as template; (b) 6F/Mwith AmpliTaq DNA Polymerase (2.5U), 1X buffer, 50 μM of each dNTP, 50pmol IM-6F (SEQ ID NO: 21), 50 pmol M-PCR (SEQ ID NO: 27) and 281 ng ofclone IM-6 (obtained from Example 3, Section H) as template.

The reactions were incubated at 95° C. for 105 seconds, and thenamplified with 20 cycles of 94° C. for 15 seconds, 60° C. for 30seconds, 72° C. for 60 seconds, then incubated at 720° C. for 7 minutes.The PCR products I/6R and 6F/M next were gel isolated following theprocedures as described hereinabove.

Step 2. A PCR reaction (100 μl volume) was set up with UlTma DNAPolymerase (3U) and 1X buffer along with 1.5 mM MgCl₂, 40 μM of eachdNTP, 50 pmol of I-PCR (SEQ ID NO: 26), 50 pmol M-PCR (SEQ ID NO: 27),50 ng I/6R, and ˜20 ng 6F/M. The reaction was incubated at 95° C. for105 seconds, and then it was amplified with 20 cycles of 94° C. for 15seconds, 55° C. for 30 seconds, 72° C. for 60 seconds, followed byincubation at 72° C. for 7 minutes. The PCR product was processed on aCentri-sep column (Princeton Separations) following the manufacturer'sinstructions.

J. Construction of pGO-11PL/DH5α.

FIGS. 4A through 4F show a diagrammatic representation of the stepsinvolved in construction of pGO-11PL/DH5α. pGO-11PL/DH5α encodes therecombinant protein pGO-11PL. FIG. 9 presents the amino acid sequence ofthe pGO-11PL recombinant protein (SEQ ID NO: 52). This protein consistsof an N-terminal methionine, 45 amino acids of env gp120 (HIV-1 group O,HAM112 isolate), and 327 amino acids of env gp41 (HIV-1 group O, HAM112isolate). pGO-11PL/DH5α was constructed as follows.

The final PCR product from Example 3, Section I and pGO-9PL vector(miniprep H5 from Example 3, section F) were digested sequentially withAge I and Bam HI. The digested pGO-9PL was then treated with calfintestinal alkaline phosphatase (BRL Life Technologies) for 15 minutesat 37° C., phenol/chloroform extracted, and precipitated with NaOAc andEtOH. The vector (pGO-9PL) was subsequently gel-isolated. The digestedpGO-9PL and the digested PCR product were ligated, and the ligationproduct was used to transform DH5α competent cells. Colonies wererestreaked for isolation. Clone pGO11-4 then was identified andrestreaked for isolation. An overnight culture of pGO11-4 was preparedin order to generate frozen stocks and perform miniprep DNA forsequencing. Clone pGO11-4 was sequenced with the followingoligonucleotide primers: pKRREcoR1 Forward (SEQ ID NO: 38), pKRRBamHIReverse (SEQ ID NO: 39), 41sy-1C (SEQ ID NO: 40), 41sy-2 (SEQ ID NO:41), 41sy-3 (SEQ ID NO: 42), 41sy-4 (SEQ ID NO: 23), 41sy-5B (SEQ ID NO:43), 41sy-5C (SEQ ID NO: 36) and 41sy-6B (SEQ ID NO: 37). Based on thesequencing results, this clone was designated as pGO-11PL/DH5α (SEQ IDNO: 51 presents the nucleotide sequence of the coding region, and SEQ IDNO: 52 presents the amino acid sequence of coding region).

K. Construction of pGO-11CKS/XL1.

FIGS. 4A through 4G show a diagrammatic representation of the stepsinvolved in construction of pGO-11CKS/XL1. pGO-11CKS/XL1 encodes therecombinant protein pGO-11CKS. FIG. 10 shows the amino sequence of thepGO-11CKS recombinant protein (SEQ ID NO: 54). This protein consists of246 amino acids of CKS and polylinker followed by 45 amino acids of envgp120 (HIV-1 group O, HAM112 isolate), and 327 amino acids of env gp41(HIV-1 group O, HAM112 isolate). pGO-11CKS/XL1 was constructed asfollows.

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U)and 1X buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 pmol ofOsyn-5'CKS (SEQ ID NO: 25), 50 pmol Osyn-M (SEQ ID NO: 14), and 1 ngpGO11-4 (obtained from Example 3, Section J) as template. The reactionwas incubated at 94° C. for 105 seconds, and then amplified with 20cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 120seconds, followed by incubation at 72° C. for 7 minutes. TheOsyn-5'CKS/Osyn-M PCR product was gel isolated. Next, theOsyn-5'CKS/Osyn-M PCR product and the vector pJO200 were EcoR I+Bam HIdigested. The digested pJO200 vector was gel isolated. Overnight (16°C.) ligations were set up with the digested PCR product. XL1-Bluesupercompetent cells were transformed with the ligation and plated onLB+ampicillin plates supplemented with 20 mM glucose. Colonies wererestreaked for isolation on the same plates. An overnight culture (LBmedium+100 μg/ml carbenicillin+20 mM glucose) of clone pGO-11CKS clonecandidate 2 then was set up. Frozen stocks (0.5 ml 80% glycerol+0.5 mlovernight culture) were made as well as miniprep DNA for sequencing. Thefollowing oligonucleotides were used as primers for sequence analysis:CKS-1 (SEQ ID NO: 30), CKS-2 (SEQ ID NO: 31), CKS-3 (SEQ ID NO: 32),CKS-4 (SEQ ID NO: 33), 43461 (SEQ ID NO: 2), 43285 (SEQ ID NO: 1),41sy-1B (SEQ ID NO: 29), 41sy-2B (SEQ ID NO: 34), 41sy-3B (SEQ ID NO:35), 41sy-4 (SEQ ID NO: 23), 41sy-5C (SEQ ID NO: 36), 41sy-6B (SEQ IDNO: 37), CKS176.1 (SEQ ID NO: 19), CKS3583 (SEQ ID NO: 20), and pTB-S8(SEQ ID NO: 28). pGO-11CKS clone #2 was designated as pGO-11CKS/XL1. SEQID NO: 53 presents the nucleotide sequence of the coding region ofpGO-11CKS/XL1, and SEQ ID NO: 54 presents the amino acid sequence of thecoding region of pGO-11CKS/XL1.

Example 4 Construction of pHIV210/XL1-Blue

FIG. 11 presents the amino acid sequence of the pHIV-210 recombinantprotein (SEQ ID NO: 55). This protein consists of 247 amino acids ofCKS/linker sequences, 60 amino acids from env gp120 (#432-491; HIV-2isolate D194.10), and 159 amino acids of env gp36 (#492-650; HIV-2isolate D194.10). The construction of pHIV210/XL1-Blue was accomplishedas follows.

The genomic DNA of HIV-2 isolate D194.10 H. Kuhnel et al., Nucleic AcidsResearch 18: 6142 (1990)! was cloned into the EMBL3 lambda cloningvector. See H. Kuhnel et al., Proc. Nat'l. Acad. Sci. USA 86: 2383-2387(1989), and H. Kuhnel et al., Nucleic Acids Research 18: 6142 (1990),incorporated herein by reference. The lambda clone containing D194.10(lambda A10) was received from Diagen Corporation, Dusseldorf, Germany.A PCR reaction (100 μl volume) was set up using AmpliTaq DNA polymerase(3.75 units), 200 μM each dATP, dCTP, dGTP, and dTTP, 0.5 μg primer 3634(SEQ ID NO:88; annealing to positions 7437-7455 on the HIV-2 isolateD194.10 (EMBL accession #X52223), 0.5 μg primer 3636 (SEQ ID NO: 89,annealing to positions 8095-8077), 1X PCR buffer, and 5 μl of the lambdaA10DNA diluted 1:50. The reaction was incubated 5 minutes at 94° C. thenamplified with 35 cycles of 94° C. for 1 minute, 45° C. for 1 minute,72° C. for 2 minutes; followed by an incubation at 72° C. for 5 minutes.The PCR reaction was extracted with phenol/chloroform (BoehringerMannheim Corporation, Indianapolis, Ind.) and the DNA was ethanol (AAPERAlcohol & Chemical Company, Shelbyville, Ky.) precipitated. The DNA wasdigested with EcoRI+Bam HI and gel purified on an 1.5% agarose gel(SeaKem GTG agarose, FMC Corporation, Rockland, Me.). The purifiedproduct was ligated into EcoRI+Bam HI digested pJO200 vector using 800units of T4 DNA ligase (New England BioLabs). XL1-Blue supercompetentcells (Stratagene) were transformed with 2 μl of the ligation asoutlined by the manufacturer and plated on LB plates supplemented withampicillin (Sigma Chemical Company). Overnight cultures were establishedby inoculating single colonies into Superbroth II media (GIBCO BRL,Grand Island, N.Y.) supplemented with 50 μg/ml ampicillin (Sigma) and 20mM glucose (Sigma). Frozen stocks were established by adding 0.3 ml of80% glycerol to 0.7 ml of overnight. After mixing stocks were stored at-70° C. Miniprep DNA was prepared from the overnight cultures using thealkaline lysis method followed by PEG precipitation. Sequence reactionswere performed with a 7-deaza-dGTP Reagent Kit with Sequenase Version2.0 (United States Biochemical Corporation, Cleveland, Ohio.) asoutlined by the manufacturer. Reactions were run on 6% acrylamide gels(GIBCO BRL Gel-Mix 6) using the IBI gel apparatus as recommended by themanufacturer. Based on sequencing results, pHIV-210 clone #7 wasdesignated as pHIV-210. The amino acid sequence of the pHIV-210 codingregion is presented as SEQ ID NO: 55.

Example 5 Growth And Induction of E. coli Strains with HIV-1 Group ORecombinant gp41 Antigen Construct

Overnight seed cultures of pGO-9CKS/XL1 were prepared in 500 ml sterileExcell Terrific Broth (available from Sigma Chemical Corp., St. LouisMo.) supplemented with 100 μg/ml sodium ampicillin, and placed in ashaking orbital incubator at 32° C. or 37° C. One hundred milliliter(100 μl) inoculums from seed cultures were transferred to flaskscontaining 1 liter sterile Excell Terrific Broth supplemented with 100μg/ml sodium ampicillin. Cultures were either (1) incubated at 37° C.until the culture(s) reached mid-logarithmic growth and then inducedwith 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C.Alternatively, the pL constructs were incubated at 32° C. until theculture(s) reached mid-logarithmic growth and then induced for 3 hoursby shifting the temperature of the culture(s) to 42° C. After theinduction period, cells were pelleted by centrifugation and harvestedfollowing standard procedures. Pelleted cells were stored at -70° C.until further processed.

Example 6 Isolation and solubilization of HIV-1 Group 0 Recombinant gp41Antigen Produced as Insoluble Inclusion Bodies in E. coli

Frozen cells obtained from Example 5 were resuspended by homogenizationin cold lysis buffer comprising 50 mM Tris pH 8, 10 mM Na EDTA, 150 mMNaCl, 8% (w/v) sucrose, 5% Triton X-100® (v/v), 1 mM PMSF and 1 μMpepstatin A. Lysozyme was added to the homogenates at a concentration of1.3 mg per gram of cells processed, and the resultant mixture wasincubated for 30 minutes on ice to lyse the cells. Inclusion bodies wereseparated from soluble proteins by centrifugation. These pelletedinclusion bodies were washed and pelleted sequentially in (1) LysisBuffer; (2) 10 mM Na EDTA pH 8, 30% (w/v) sucrose; and (3) water. Thewashed inclusion bodies were resuspended in 50 mM Tris pH 8, 10 mM NaEDTA, 150 mM NaCl and 3 M urea, and incubated on ice for 1 hour. Theinclusion bodies then were separated from the solubilized proteins bycentrifugation. The pelleted inclusion bodies were fully solubilized in7 M guanidine-HCl, 50 mM Tris pH 8, 0.1% (v/v) beta-mercaptoethanol(BME) overnight at 4° C. The solubilized recombinant antigens wereclarified by centrifugation, passed through a 0.2 μm filter and storedat ≦-20° C. until purified by chromatography.

Example 7 Purification of Recombinant HIV-1 Group O gp41 Antigen byChromatography

Solubilized HIV-1 Group 0 recombinant gp41 antigens obtained fromExample 6 were purified by a two step method, as follows. Guanidine-HClextracts of insoluble antigens were purified by size exclusionchromatography on a Sephacryl S-300 column equilibrated with 50 mM TrispH 8, 8 M Urea and 0.1% BME (v/v). SDS-polyacrylamide electrophoresiswas used to analyze fractions. Fractions containing the recombinant gp41antigen were pooled and then concentrated by ultrafiltration. Therecombinant antigen concentrate was treated with 4% SDS (w/v) and 5% BME(w/v) at room temperature for 3 hours. SDS treated antigen was furtherpurified by size exclusion chromatography on a Sephacryl S-300 columnequilibrated with 25 mM Tris pH 8, 0.15 M NaCl, 0.1% v/v BME, 0.1% SDS(w/v). SDS-polyacrylamide electrophoresis was used to analyze thefractions. Fractions containing purified recombinant antigen werepooled, passed through a 0.2 μm filter and stored at -70° C.

Example 8 Preparation of HIV-1 Group M Antigen

Cells containing the plasmid pTB319 were grown and induced as describedin Example 5. Cells were lysed and inclusion bodies were processedessentially as described in Example 5 of U.S. Pat. No: 5,124,255,incorporated herein by reference. The pellet material was subsequentlysolubilized in SDS, Phosphate, pH 6.8 and then subjected tochromatography on an S-300 column.

Example 9 Preparation of HIV-2 Antigen

pHIV-210/XL1-Blue cells (Example 4, hereinabove) were grown and inducedas described in Example 5. Cells were lysed with a buffer containingphosphate, MgCl₂, Na EDTA, Triton X-100® pH 7.4 supplemented withBenzonase, Lysozyme, and PMSF. Inclusion bodies were separated fromsoluble proteins by centrifugation. The pellet was washed sequentiallywith: distilled H₂ O; Triton X-100®, deoxycholate, NaCl, Phosphate pH7.0; 50 mM Phosphate, pH 7.0; urea, SDS in phosphate, pH 7.0+BME.Proteins were solubilized in SDS, phosphate, pH 7.0 and BME thensubjected to chromatography on an S300 column. Example 10. One StepImmunochromatographic Assay For Simultaneous Detection andDifferentiation of HIV-1 group M. HIV-1 group O and HIV-2

A. Reagent preparation

1. A selenium (Se) colloid suspension was prepared substantially asfollows: SeO₂ was dissolved in water to a concentration of 0.0625 gm/ml.Ascorbate then was dissolved in water to a concentration of 0.32 gm/mland heated in a 70° C. water bath for 24 hours. The ascorbate solutionthen was diluted to 0.0065 gm/ml in water. The SeO₂ solution was quicklyadded to the diluted ascorbate solution and incubated at 42° C.Incubation was ended after a minimum of 42 hours when the absorbancemaximum exceeded 30 at a wavelength between 542 nm and 588 nm. Thecolloid suspension was cooled to 2-8° C., then stored. Selenium colloidsuspension is available from Abbott Laboratories, Abbott Park, Ill.(Code 25001).

2. Selenium colloid/antibody conjugates were prepared as follows. Theselenium colloid suspension was concentrated to an absorbance of 25 (OD500-570) in distilled water. Then, 1 M MOPS was added to a finalconcentration of 10 mM pH 7.2. Goat antibodies specific for human IgG Fcregion (or other species of antibody specific for human IgG Fc region)were diluted to a concentration of 0.75 mg/ml with 50 mM Phosphatebuffer, and the resultant antibody preparation then was added withmixing to the selenium colloid suspension prepared as describedhereinabove, to a final antibody concentration of 75 μg/ml. Stirring wascontinued for 40 minutes. Then, 1% (by weight) bovine serum albumin(BSA) was added to the solution, and the selenium colloid/antibodyconjugate solution was stirred for an additional 15 minutes andcentrifuged at 5000×g for 90 minutes. Following this, 90% of thesupernatant was removed, and the pellet was resuspended with theremaining supernatant. Immediately prior to coating this selenium-IgGconjugate to a glass fiber pad, it was diluted 1:10 with conjugatediluent (1% by weight! casein, 0.1% weight! Triton X-405®, and 50 mMTris, pH 8.2).

3. Procedural control reagent was prepared as a mixture of HIV-1 (groupM), HIV-1 (group O), and HIV-2 positive sera, and is utilized on aseparate strip device as a positive control of the assay.

4. Negative control reagent used was normal human utilized on a separatetest device as a negative control of the assay.

B. Application pad preparation

The application pad material comprises resin bonded glass fiber paper(Lydall). Approximately 0.1 ml of the prepared conjugate (described inpreceding paragraph 2) is applied to the application pad.

C. Chromatographic Material Preparation

All reagents are applied to a nitrocellulose membrane by charge anddeflect reagent jetting. The nitrocellulose is supported by a MYLAR®membrane that is coated with a pressure sensitive adhesive.

The test sample capture reagents were prepared by (a) diluting thespecific antigen prepared as described hereinabove to a concentration of0.5 mg/ml in jetting diluent (100 mM Tris, pH 7.6 with 1% sucrose (byweight), 0.9% NaCl and 5 μg/ml fluorescein) for HIV-1 group O capturereagent (pGO-9/CKS, SEQ ID NO: 50), (b) for HIV-1 group M, subgroup Bcapture reagent (pTB319, SEQ ID NO: 56), and (c) for HIV-2 capturereagent (pHIV-210, SEQ ID NO: 55). 0.098 μl of a first capture reagent(reagent HIV-1 group M subgroup B; SEQ ID NO: 56) was applied to thestrip at the designated capture location and constituted one patientcapture site. Likewise, 0.098 μl of a second capture reagent (reagentHIV-1 group O; SEQ ID NO: 50) was applied to the strip at the designatedcapture location and constituted one patient capture site, and 0.098 μlof a third capture reagent (reagent HIV-2; SEQ ID NO: 55) was applied tothe strip at the designated capture location and constituted one patientcapture site.

D. Rapid assay for the presence of antibodies to HIV

A rapid assay for the presence of antibodies to HIV in test samplesserum, whole blood, saliva, and urine samples was performed as follows.In a 1.5 ml Eppendorf tube, 5 μl of serum and 600 μl of sample elutionbuffer (SEB) (containing 50 mM Tris, 1% BSA (w/v), 0.4% Triton X-405®(v/v), 1.5% Casein (w/v), 3% Bovine IgG (w/v), 4% E. coli lysate (v/v),pH 8.2!) was mixed. Four drops of this mixture was applied to the samplewell of the STAR housing. Next, 1 μl of serum or whole blood was addedto 100 μl of SEB in a well of a microtiter plate, and the nitrocellulosestrip was added in the well. Following this, 1 μl of serum or wholeblood was spotted in the test device of the invention's sample welldirectly and 4 drops of SEB was added. When testing saliva, 50 or 75 μlof saliva was added to 50 μl or 25 μl of SEB, respectively, in a well ofa microtiter plate, and the nitrocellulose test strip then was added tothe well. When testing urine, 50 μl of urine was added to 50 ul of SEBin a well of a microtiter plate, and the nitrocellulose test strip wasadded in the well. Alternatively, 100 μl of urine was used in the wellof a microtiter plate, and the nitrocellulose test strip was added,without using SEB.

The IgG in the sample was bound by the selenium-goat anti-human IgGcolloid in the conjugate pad, and the complexes were chromatographedalong the length of the nitrocellulose membrane test strips on which thethree recombinant antigens pGO-9 CKS SEQ ID NO: 50), pTB319 (HIV-1 groupM (subgroup B), SEQ ID NO: 56) and pHIV210 (HIV-2, SEQ ID NO: 55)previously were applied at a concentration of 1 mg/ml using a biodotmachine, which provided positive displacement dispensing using precisedrop sizes. The test device then was incubated at room temperature fortwo minutes, and the results were read visually.

E. Spiked Whole Blood Assay

In a 1.5 ml Eppendorf tube, the equivalent of 1 μl blood from eitherconfirmed positive HIV-1 group O, HIV-1 group M or HIV-2, or confirmednegative for HIV-1 group O, HIV-1 group M or HIV-2 whole blood testsample was added to 5 μl of a confirmed negative HIV-1 group O, HIV-1group M or HIV-2 serum along with 100 μl of SEB, and mixed. This mixturewas applied to the sample well of the test device of the invention.

The IgG in the sample was bound by the selenium-goat anti-human IgGcolloid in the conjugate pad, and the complexes were chromatographedalong the length of the nitrocellulose membrane test strips on which thethree recombinant antigens pGO-9 CKS SEQ ID NO: 50), pTB319 (HIV-1 groupM (subgroup B), SEQ ID NO: 56) and pHIV210 (HIV-2, SEQ ID NO: 55)previously were applied at a concentration of 1 mg/ml using a biodotmachine, which provided positive displacement dispensing using precisedrop sizes. The test device then was incubated at room temperature fortwo minutes, and the results were read visually.

F. Results

If antibody to antigen 1 was present in the test sample, a visiblereaction was indicated in the capture zone area of antigen 1 and in theassay completion zone, and not in the zones of antigen 2 or antigen 3.If antibody to antigen 2 was present in the test sample, a visiblereaction was indicated in the capture zone area of antigen 2 and in theassay completion zone, and not in the zones of antigen 1 or antigen 3.If antibody to antigen 3 was present in the test sample, a visiblereaction was indicated in the capture zone area of antigen 3 and in theassay completion zone, and not in the zones of antigen 1 or antigen 2.Also, a negative control should be non-reactive (show no visiblereaction) in the zones of antigen 1, antigen 2 and antigen 3, but shouldbe reactive in the assay completion zone. A positive control (knownreactive antibody to antigen 1, 2 and/or 3) should be reactive in thezone of the appropriate antigen to which it specifically binds in anantigen/antibody reaction. A result was considered invalid when apositive reaction occurred in one of the antigen capture zones but notin the assay completion zone, and the test was repeated.

(i) Assaying for antibodies in Blood, Urine and Saliva. The blood,urine, and saliva of three patients (identified by patient numbers 0109,4068, and 4475) were tested on nitrocellulose solid phase devices of theinvention as described herein and following the assay protocol as setforth hereinabove. Each blood and urine test sample of each patient0109, 4068 and 4475 was reactive with antigen 1 (pTB319; SEQ ID NO 56).The saliva test sample of patients 4068 and 4475 also were reactive withantigen 1, while patient 0109's saliva test sample was non-reactive inthe test device of the invention. The saliva test sample of patient 0109was later retested by a standard EIA and confirmed non-reactive forantibodies to HIV-1 gp41, indicating that the results obtained for thesaliva test sample of patient 0109 were valid.

(ii) Assaying Negative Samples for HIV antibodies. FIG. 14 is aphotograph of four test devices and shows the results obtained testingtwo negative sera and two negative whole blood test samples, each spikedwith the same two negative sera. Samples contained no antibodiesspecific for the relevant antigens and the test samples were negativeafter assay on the test (i.e. no reactivity, as indicated by no visiblebar signifying a reaction in either position O, M or 2. Test sample waspresent in each test device, as indicated by the positive reaction barin the test sample reactivity zone.

(iii) Assaying for HIV-1 group M antibody. FIG. 15 is a photograph of 10test devices and shows the results obtained testing five HIV-1 group Msera and five whole blood samples spiked with the HIV-1 group M positivesera. As can be seen in FIG. 15, HIV-1 group M samples containedantibodies specific for HIV-1 group M antigen (pTB319: middle zone) anddeveloped a reaction line at the HIV-1 group M antigen zone, and visiblereaction lines can be seen in the assay completion zone labeled "M" ofnine out of 10 test devices. Although a band was present in oneparticular test device in the capture zone for HIV-1 group M antibody ,test sample did not to the assay completion zone and thus, the assayneeded to be repeated for this particular sample. Note that nocross-reactivity was observed with the capture reagents for HIV group Oand HIV-2.

(iv) Assaying for HIV-1 group O antibodies. FIG. 16 is a photograph offour test devices, showing the results obtained when testing twoconfirmed positive HIV-1 group O sera and two whole blood test samplesspiked with HIV-1 group O sera. As can be seen in FIG. 16, HIV-1 group Osamples contained antibodies specific for HIV-1 group O antigen asindicated by the positive bar result in the HIV-1 group O antigencapture zone area (lowest zone, indicated as "O"), visible reactionlines can be seen in the assay completion zone of each device, and nocross-reaction with HIV-1 group M or HIV-2 capture antigens (no visiblebar) was observed.

(v) Assaying for HIV-2 Antibodies. FIG. 17 is a photograph of 10 testdevices showing the results obtained with five HIV-2 confirmed positivesera (five test devices to the left) and whole blood spiked with the 5HIV-2 sera (five test devices to the right). As can be seen from FIG.17, HIV-2 samples contained antibodies specific for HIV-2 antigen(pHIV210, upper zone, indicated by "2") as shown by the reaction bar atthe HIV-2 antigen zone. No reaction was observed with these test samplesand HIV-1 group O antigen or HIV-1 group M antigen, and visible reactionlines can be seen in the assay completion zone of each device.

(vi) Assaying HIV-1 group M. HIV-1 group O. HIV-2 and Negative Samples.FIG. 18 is a photograph of four test devices, in which (from left toright) a negative test sample, an HIV-1 group M positive test sample, anHIV-1 group O positive test sample, and an HIV-2 positive test samplewere tested individually. As can be seen from FIG. 18, the negative testserum did not react with any antigen in the antigen capture zone, whilethe HIV-1 group M positive test sample was reactive only with the HIV-1group M antigen, the HIV-1 group O positive test sample was reactiveonly with the HIV-1 group O antigen, and the HIV-2 positive test samplewas reactive only with the HIV-2 antigen, and visible reaction lines canbe seen in the assay completion zone of each device.

The five HIV-1 group M and the two HIV-1 group O test samples used wereconfirmed seropositive samples which previously had been tested usingAbbott's 3A77 EIA and has been PCR amplified, sequenced and subtypedbased on phylogenetic analysis. The five HIV-2 samples used wereseropositive using Abbott's 3A77 EIA and were confirmed as HIV-2 samplesby an HIV-2 Western blot test (Sanofi).

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 89    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    # 19               TCC    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    # 20               ACCC    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 114 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - CATGATCGGT GGTGACATGA AAGACATCTG GCGTAACGAA CTGTTCAAAT AC - #AAAGTTGT      60    - TCGTGTTAAA CCGTTCTCTG TTGCTCCGAC CCCGATCGCT CGTCCGGTTA TC - #GG     114    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 111 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - GCAGGTTCCA CTATGGGTGC TGCAGCTACC GCTCTGACCG TACAGACCCA CT - #CTGTTATC      60    #            111AGCAGCA CGACAACCTG CTGCGTGCAA TCCAGGCACA G    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 110 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - AGCTGCTGGT TCTGGATCAG GGTTTCCAGT GCCAGCAGAC GAGCACGCAG CT - #GACGGATA      60    #             110ACGCAG CAGTTCCTGC TGTGCCTGGA TTGCACGCAG    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 111 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - CTGATCCAGA ACCAGCAGCT GCTGAACCTG TGGGGCTGCA AAGGTCGTCT GA - #TCTGCTAC      60    #            111GGAACGA AACCTGGCGT AACACCACCA ACATCAACCA G    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 117 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - CTGAACCTGA GCTTTCTGGA TTTCTTCGTA GATGGTGGAA GAAACGTTGT CG - #ATCTGCTG      60    - GTCCCATTCC TGCCAGGTCA GGTTACCCCA GATCTGGTTG ATGTTGGTGG TG - #TTACG     117    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 101 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - TCCAGAAAGC TCAGGTTCAG CAGGAACAGA ACGAAAAAAA ACTGCTGGAA CT - #GGACGAAT      60    #  101             CTGG CTGGACATCA CCAAATGGCT G    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 114 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - ACCTTCACCG GTACGACCCG GAGTTTCAGC TTCAGACTGC TGACGGGTCG GG - #ATCTGCAG      60    - GGACAGCGGC TGGTAGCCCT GACGGATGTT ACGCAGCCAT TTGGTGATGT CC - #AG     114    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 107 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - CGGGTCGTAC CGGTGAAGGT GGTGGTGACG AAGGCCGTCC GCGTCTGATC CC - #GTCTCCGC      60    #               107GCTG TACACCGACC TGCGTACCAT CATCCTG    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 31 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #          31      ATCG GTGGTGACAT G    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 109 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - GTCTGTGGAT TCTGGGTCAG AAAATCATCG ACGCTTGCCG TATCTGCGCT GC - #TGTTATCC      60    #              109AACTG CAGAAATCCG CTACCTCCCT GATCGACAC    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 114 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    - GCGAACACGA CGCGGGATGT TCAGGATACC ACGACCCAGA CGCTGGATAC CA - #CGGATGAT      60    - GTCGTCAGTC CAGTTAGCAA CTGCAACAGC GAAGGTGTCG ATCAGGGAGG TA - #GC     114    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 60 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    - ATAGTAGGAT CCTATTACAG CAGAGAGCGT TCGAAGCCCT GGCGAACACG AC - #GCGGGATG      60    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 43 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    # 43               ATTC ACCGGTACGA CCCGGAGTTT CAG    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 38 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #     38           ACAG CCATTTGGTG ATGTCCAG    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 106 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - GCACCCATAG TGGAACCTGC TGCAGACAGA ACGCCCAGGA ACAGCATACC CA - #GACCTACA      60    #                106GGT GCCAGTACCG ATAACCGGAC GAGCGA    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 108 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - CTGACCCAGA ATCCACAGAC CCAGACGCAG GTGAGAGATA ACAGTCTGAG TA - #CCAGAGAT      60    #               108TGGT AGGACCACAG GATGATGGTA CGCAGGTC    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    #              26  TGGT ACGGCG    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    # 19               TCC    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    #              26  CGAC ACCTTC    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    #              26  GAGG TAGCGG    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    # 19               CAC    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 64 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    - CTACAAGAAT TCCATGATCG GTGGTGACAT GAAAGACATC TGGCGTAACG AA - #CTGTTCAA      60    #             64    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 34 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    #        34        GGTG GTGACATGAA AGAC    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    # 20               AGGT    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    #                23ACAG CAG    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    # 19               ATC    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    # 19               GAC    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    #21                AGCT C    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    # 20               CGCA    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    # 19               GTG    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    #  18              CA    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    # 19               GTC    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    # 20               GAAC    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    # 20               CTGG    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    # 20               ACAG    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    #                 22ATT GG    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    #21                CAAC C    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    #21                ATCG T    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    #  18              GC    - (2) INFORMATION FOR SEQ ID NO:42:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    #  18              CG    - (2) INFORMATION FOR SEQ ID NO:43:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    #  18              TC    - (2) INFORMATION FOR SEQ ID NO:44:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    #   17             C    - (2) INFORMATION FOR SEQ ID NO:45:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    # 20               AAGC    - (2) INFORMATION FOR SEQ ID NO:46:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    # 20               AGGG    - (2) INFORMATION FOR SEQ ID NO:47:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 741 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    - ATGATCGGTG GTGACATGAA AGACATCTGG CGTAACGAAC TGTTCAAATA CA - #AAGTTGTT      60    - CGTGTTAAAC CGTTCTCTGT TGCTCCGACC CCGATCGCTC GTCCGGTTAT CG - #GTACTGGC     120    - ACCCACCGTG AAAAACGTGC TGTAGGTCTG GGTATGCTGT TCCTGGGCGT TC - #TGTCTGCA     180    - GCAGGTTCCA CTATGGGTGC TGCAGCTACC GCTCTGACCG TACAGACCCA CT - #CTGTTATC     240    - AAAGGTATCG TACAGCAGCA GGACAACCTG CTGCGTGCAA TCCAGGCACA GC - #AGGAACTG     300    - CTGCGTCTGT CTGTATGGGG TATCCGTCAG CTGCGTGCTC GTCTGCTGGC AC - #TGGAAACC     360    - CTGATCCAGA ACCAGCAGCT GCTGAACCTG TGGGGCTGCA AAGGTCGTCT GA - #TCTGCTAC     420    - ACCTCCGTTA AATGGAACGA AACCTGGCGT AACACCACCA ACATCAACCA GA - #TCTGGGGT     480    - AACCTGACCT GGCAGGAATG GGACCAGCAG ATCGACAACG TTTCTTCCAC CA - #TCTACGAA     540    - GAAATCCAGA AAGCTCAGGT TCAGCAGGAA CAGAACGAAA AAAAACTGCT GG - #AACTGGAC     600    - GAATGGGCTT CTCTGTGGAA CTGGCTGGAC ATCACCAAAT GGCTGCGTAA CA - #TCCGTCAG     660    - GGCTACCAGC CGCTGTCCCT GCAGATCCCG ACCCGTCAGC AGTCTGAAGC TG - #AAACTCCG     720    #                 741TA G    - (2) INFORMATION FOR SEQ ID NO:48:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 245 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    - Met Ile Gly Gly Asp Met Lys Asp Ile Trp Ar - #g Asn Glu Leu Phe Lys    #                 15    - Tyr Lys Val Val Arg Val Lys Pro Phe Ser Va - #l Ala Pro Thr Pro Ile    #             30    - Ala Arg Pro Val Ile Gly Thr Gly Thr His Ar - #g Glu Lys Arg Ala Val    #         45    - Gly Leu Gly Met Leu Phe Leu Gly Val Leu Se - #r Ala Ala Gly Ser Thr    #     60    - Met Gly Ala Ala Ala Thr Ala Leu Thr Val Gl - #n Thr His Ser Val Ile    # 80    - Lys Gly Ile Val Gln Gln Gln Asp Asn Leu Le - #u Arg Ala Ile Gln Ala    #                 95    - Gln Gln Glu Leu Leu Arg Leu Ser Val Trp Gl - #y Ile Arg Gln Leu Arg    #           110    - Ala Arg Leu Leu Ala Leu Glu Thr Leu Ile Gl - #n Asn Gln Gln Leu Leu    #       125    - Asn Leu Trp Gly Cys Lys Gly Arg Leu Ile Cy - #s Tyr Thr Ser Val Lys    #   140    - Trp Asn Glu Thr Trp Arg Asn Thr Thr Asn Il - #e Asn Gln Ile Trp Gly    145                 1 - #50                 1 - #55                 1 -    #60    - Asn Leu Thr Trp Gln Glu Trp Asp Gln Gln Il - #e Asp Asn Val Ser Ser    #               175    - Thr Ile Tyr Glu Glu Ile Gln Lys Ala Gln Va - #l Gln Gln Glu Gln Asn    #           190    - Glu Lys Lys Leu Leu Glu Leu Asp Glu Trp Al - #a Ser Leu Trp Asn Trp    #       205    - Leu Asp Ile Thr Lys Trp Leu Arg Asn Ile Ar - #g Gln Gly Tyr Gln Pro    #   220    - Leu Ser Leu Gln Ile Pro Thr Arg Gln Gln Se - #r Glu Ala Glu Thr Pro    225                 2 - #30                 2 - #35                 2 -    #40    - Gly Arg Thr Gly Glu                    245    - (2) INFORMATION FOR SEQ ID NO:49:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1476 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    - ATGAGTTTTG TGGTCATTAT TCCCGCGCGC TACGCGTCGA CGCGTCTGCC CG - #GTAAACCA      60    - TTGGTTGATA TTAACGGCAA ACCCATGATT GTTCATGTTC TTGAACGCGC GC - #GTGAATCA     120    - GGTGCCGAGC GCATCATCGT GGCAACCGAT CATGAGGATG TTGCCCGCGC CG - #TTGAAGCC     180    - GCTGGCGGTG AAGTATGTAT GACGCGCGCC GATCATCAGT CAGGAACAGA AC - #GTCTGGCG     240    - GAAGTTGTCG AAAAATGCGC ATTCAGCGAC GACACGGTGA TCGTTAATGT GC - #AGGGTGAT     300    - GAACCGATGA TCCCTGCGAC AATCATTCGT CAGGTTGCTG ATAACCTCGC TC - #AGCGTCAG     360    - GTGGGTATGA CGACTCTGGC GGTGCCAATC CACAATGCGG AAGAAGCGTT TA - #ACCCGAAT     420    - GCGGTGAAAG TGGTTCTCGA CGCTGAAGGG TATGCACTGT ACTTCTCTCG CG - #CCACCATT     480    - CCTTGGGATC GTGATCGTTT TGCAGAAGGC CTTGAAACCG TTGGCGATAA CT - #TCCTGCGT     540    - CATCTTGGTA TTTATGGCTA CCGTGCAGGC TTTATCCGTC GTTACGTCAA CT - #GGCAGCCA     600    - AGTCCGTTAG AACACATCGA AATGTTAGAG CAGCTTCGTG TTCTGTGGTA CG - #GCGAAAAA     660    - ATCCATGTTG CTGTTGCTCA GGAAGTTCCT GGCACAGGTG TGGATACCCC TG - #AAGATCTC     720    - GACCCGTCGA CGAATTCTAT CGGTGGTGAC ATGAAAGACA TCTGGCGTAA CG - #AACTGTTC     780    - AAATACAAAG TTGTTCGTGT TAAACCGTTC TCTGTTGCTC CGACCCCGAT CG - #CTCGTCCG     840    - GTTATCGGTA CTGGCACCCA CCGTGAAAAA CGTGCTGTAG GTCTGGGTAT GC - #TGTTCCTG     900    - GGCGTTCTGT CTGCAGCAGG TTCCACTATG GGTGCTGCAG CTACCGCTCT GA - #CCGTACAG     960    - ACCCACTCTG TTATCAAAGG TATCGTACAG CAGCAGGACA ACCTGCTGCG TG - #CAATCCAG    1020    - GCACAGCAGG AACTGCTGCG TCTGTCTGTA TGGGGTATCC GTCAGCTGCG TG - #CTCGTCTG    1080    - CTGGCACTGG AAACCCTGAT CCAGAACCAG CAGCTGCTGA ACCTGTGGGG CT - #GCAAAGGT    1140    - CGTCTGATCT GCTACACCTC CGTTAAATGG AACGAAACCT GGCGTAACAC CA - #CCAACATC    1200    - AACCAGATCT GGGGTAACCT GACCTGGCAG GAATGGGACC AGCAGATCGA CA - #ACGTTTCT    1260    - TCCACCATCT ACGAAGAAAT CCAGAAAGCT CAGGTTCAGC AGGAACAGAA CG - #AAAAAAAA    1320    - CTGCTGGAAC TGGACGAATG GGCTTCTCTG TGGAACTGGC TGGACATCAC CA - #AATGGCTG    1380    - CGTAACATCC GTCAGGGCTA CCAGCCGCTG TCCCTGCAGA TCCCGACCCG TC - #AGCAGTCT    1440    #     1476         GTCG TACCGGTGAA TAATAG    - (2) INFORMATION FOR SEQ ID NO:50:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 490 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    - Met Ser Phe Val Val Ile Ile Pro Ala Arg Ty - #r Ala Ser Thr Arg Leu    #                 15    - Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly - #s Pro Met Ile Val His    #             30    - Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Gl - #u Arg Ile Ile Val Ala    #         45    - Thr Asp His Glu Asp Val Ala Arg Ala Val Gl - #u Ala Ala Gly Gly Glu    #     60    - Val Cys Met Thr Arg Ala Asp His Gln Ser Gl - #y Thr Glu Arg Leu Ala    # 80    - Glu Val Val Glu Lys Cys Ala Phe Ser Asp As - #p Thr Val Ile Val Asn    #                 95    - Val Gln Gly Asp Glu Pro Met Ile Pro Ala Th - #r Ile Ile Arg Gln Val    #           110    - Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Me - #t Thr Thr Leu Ala Val    #       125    - Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pr - #o Asn Ala Val Lys Val    #   140    - Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Ph - #e Ser Arg Ala Thr Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Le - #u Glu Thr Val Gly Asp    #               175    - Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Ty - #r Arg Ala Gly Phe Ile    #           190    - Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Le - #u Glu His Ile Glu Met    #       205    - Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Gl - #u Lys Ile His Val Ala    #   220    - Val Ala Gln Glu Val Pro Gly Thr Gly Val As - #p Thr Pro Glu Asp Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Asp Pro Ser Thr Asn Ser Ile Gly Gly Asp Me - #t Lys Asp Ile Trp Arg    #               255    - Asn Glu Leu Phe Lys Tyr Lys Val Val Arg Va - #l Lys Pro Phe Ser Val    #           270    - Ala Pro Thr Pro Ile Ala Arg Pro Val Ile Gl - #y Thr Gly Thr His Arg    #       285    - Glu Lys Arg Ala Val Gly Leu Gly Met Leu Ph - #e Leu Gly Val Leu Ser    #   300    - Ala Ala Gly Ser Thr Met Gly Ala Ala Ala Th - #r Ala Leu Thr Val Gln    305                 3 - #10                 3 - #15                 3 -    #20    - Thr His Ser Val Ile Lys Gly Ile Val Gln Gl - #n Gln Asp Asn Leu Leu    #               335    - Arg Ala Ile Gln Ala Gln Gln Glu Leu Leu Ar - #g Leu Ser Val Trp Gly    #           350    - Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Le - #u Glu Thr Leu Ile Gln    #       365    - Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Ly - #s Gly Arg Leu Ile Cys    #   380    - Tyr Thr Ser Val Lys Trp Asn Glu Thr Trp Ar - #g Asn Thr Thr Asn Ile    385                 3 - #90                 3 - #95                 4 -    #00    - Asn Gln Ile Trp Gly Asn Leu Thr Trp Gln Gl - #u Trp Asp Gln Gln Ile    #               415    - Asp Asn Val Ser Ser Thr Ile Tyr Glu Glu Il - #e Gln Lys Ala Gln Val    #           430    - Gln Gln Glu Gln Asn Glu Lys Lys Leu Leu Gl - #u Leu Asp Glu Trp Ala    #       445    - Ser Leu Trp Asn Trp Leu Asp Ile Thr Lys Tr - #p Leu Arg Asn Ile Arg    #   460    - Gln Gly Tyr Gln Pro Leu Ser Leu Gln Ile Pr - #o Thr Arg Gln Gln Ser    465                 4 - #70                 4 - #75                 4 -    #80    - Glu Ala Glu Thr Pro Gly Arg Thr Gly Glu    #               490    - (2) INFORMATION FOR SEQ ID NO:51:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1125 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    - ATGATCGGTG GTGACATGAA AGACATCTGG CGTAACGAAC TGTTCAAATA CA - #AAGTTGTT      60    - CGTGTTAAAC CGTTCTCTGT TGCTCCGACC CCGATCGCTC GTCCGGTTAT CG - #GTACTGGC     120    - ACCCACCGTG AAAAACGTGC TGTAGGTCTG GGTATGCTGT TCCTGGGCGT TC - #TGTCTGCA     180    - GCAGGTTCCA CTATGGGTGC TGCAGCTACC GCTCTGACCG TACAGACCCA CT - #CTGTTATC     240    - AAAGGTATCG TACAGCAGCA GGACAACCTG CTGCGTGCAA TCCAGGCACA GC - #AGGAACTG     300    - CTGCGTCTGT CTGTATGGGG TATCCGTCAG CTGCGTGCTC GTCTGCTGGC AC - #TGGAAACC     360    - CTGATCCAGA ACCAGCAGCT GCTGAACCTG TGGGGCTGCA AAGGTCGTCT GA - #TCTGCTAC     420    - ACCTCCGTTA AATGGAACGA AACCTGGCGT AACACCACCA ACATCAACCA GA - #TCTGGGGT     480    - AACCTGACCT GGCAGGAATG GGACCAGCAG ATCGACAACG TTTCTTCCAC CA - #TCTACGAA     540    - GAAATCCAGA AAGCTCAGGT TCAGCAGGAA CAGAACGAAA AAAAACTGCT GG - #AACTGGAC     600    - GAATGGGCTT CTCTGTGGAA CTGGCTGGAC ATCACCAAAT GGCTGCGTAA CA - #TCCGTCAG     660    - GGCTACCAGC CGCTGTCCCT GCAGATCCCG ACCCGTCAGC AGTCTGAAGC TG - #AAACTCCG     720    - GGTCGTACCG GTGAAGGTGG TGGTGACGAA GGCCGTCCGC GTCTGATCCC GT - #CTCCGCAG     780    - GGTTTCCTGC CGCTGCTGTA CACCGACCTG CGTACCATCA TCCTGTGGTC CT - #ACCACCTG     840    - CTGTCTAACC TGATCTCTGG TACTCAGACT GTTATCTCTC ACCTGCGTCT GG - #GTCTGTGG     900    - ATTCTGGGTC AGAAAATCAT CGACGCTTGC CGTATCTGCG CTGCTGTTAT CC - #ACTACTGG     960    - CTGCAGGAAC TGCAGAAATC CGCTACCTCC CTGATCGACA CCTTCGCTGT TG - #CAGTTGCT    1020    - AACTGGACTG ACGACATCAT CCTGGGTATC CAGCGTCTGG GTCGTGGTAT CC - #TGAACATC    1080    #                1125GG CTTCGAACGC TCTCTGCTGT AATAG    - (2) INFORMATION FOR SEQ ID NO:52:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 373 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    - Met Ile Gly Gly Asp Met Lys Asp Ile Trp Ar - #g Asn Glu Leu Phe Lys    #                 15    - Tyr Lys Val Val Arg Val Lys Pro Phe Ser Va - #l Ala Pro Thr Pro Ile    #             30    - Ala Arg Pro Val Ile Gly Thr Gly Thr His Ar - #g Glu Lys Arg Ala Val    #         45    - Gly Leu Gly Met Leu Phe Leu Gly Val Leu Se - #r Ala Ala Gly Ser Thr    #     60    - Met Gly Ala Ala Ala Thr Ala Leu Thr Val Gl - #n Thr His Ser Val Ile    # 80    - Lys Gly Ile Val Gln Gln Gln Asp Asn Leu Le - #u Arg Ala Ile Gln Ala    #                 95    - Gln Gln Glu Leu Leu Arg Leu Ser Val Trp Gl - #y Ile Arg Gln Leu Arg    #           110    - Ala Arg Leu Leu Ala Leu Glu Thr Leu Ile Gl - #n Asn Gln Gln Leu Leu    #       125    - Asn Leu Trp Gly Cys Lys Gly Arg Leu Ile Cy - #s Tyr Thr Ser Val Lys    #   140    - Trp Asn Glu Thr Trp Arg Asn Thr Thr Asn Il - #e Asn Gln Ile Trp Gly    145                 1 - #50                 1 - #55                 1 -    #60    - Asn Leu Thr Trp Gln Glu Trp Asp Gln Gln Il - #e Asp Asn Val Ser Ser    #               175    - Thr Ile Tyr Glu Glu Ile Gln Lys Ala Gln Va - #l Gln Gln Glu Gln Asn    #           190    - Glu Lys Lys Leu Leu Glu Leu Asp Glu Trp Al - #a Ser Leu Trp Asn Trp    #       205    - Leu Asp Ile Thr Lys Trp Leu Arg Asn Ile Ar - #g Gln Gly Tyr Gln Pro    #   220    - Leu Ser Leu Gln Ile Pro Thr Arg Gln Gln Se - #r Glu Ala Glu Thr Pro    225                 2 - #30                 2 - #35                 2 -    #40    - Gly Arg Thr Gly Glu Gly Gly Gly Asp Glu Gl - #y Arg Pro Arg Leu Ile    #               255    - Pro Ser Pro Gln Gly Phe Leu Pro Leu Leu Ty - #r Thr Asp Leu Arg Thr    #           270    - Ile Ile Leu Trp Ser Tyr His Leu Leu Ser As - #n Leu Ile Ser Gly Thr    #       285    - Gln Thr Val Ile Ser His Leu Arg Leu Gly Le - #u Trp Ile Leu Gly Gln    #   300    - Lys Ile Ile Asp Ala Cys Arg Ile Cys Ala Al - #a Val Ile His Tyr Trp    305                 3 - #10                 3 - #15                 3 -    #20    - Leu Gln Glu Leu Gln Lys Ser Ala Thr Ser Le - #u Ile Asp Thr Phe Ala    #               335    - Val Ala Val Ala Asn Trp Thr Asp Asp Ile Il - #e Leu Gly Ile Gln Arg    #           350    - Leu Gly Arg Gly Ile Leu Asn Ile Pro Arg Ar - #g Val Arg Gln Gly Phe    #       365    - Glu Arg Ser Leu Leu        370    - (2) INFORMATION FOR SEQ ID NO:53:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1860 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    - ATGAGTTTTG TGGTCATTAT TCCCGCGCGC TACGCGTCGA CGCGTCTGCC CG - #GTAAACCA      60    - TTGGTTGATA TTAACGGCAA ACCCATGATT GTTCATGTTC TTGAACGCGC GC - #GTGAATCA     120    - GGTGCCGAGC GCATCATCGT GGCAACCGAT CATGAGGATG TTGCCCGCGC CG - #TTGAAGCC     180    - GCTGGCGGTG AAGTATGTAT GACGCGCGCC GATCATCAGT CAGGAACAGA AC - #GTCTGGCG     240    - GAAGTTGTCG AAAAATGCGC ATTCAGCGAC GACACGGTGA TCGTTAATGT GC - #AGGGTGAT     300    - GAACCGATGA TCCCTGCGAC AATCATTCGT CAGGTTGCTG ATAACCTCGC TC - #AGCGTCAG     360    - GTGGGTATGA CGACTCTGGC GGTGCCAATC CACAATGCGG AAGAAGCGTT TA - #ACCCGAAT     420    - GCGGTGAAAG TGGTTCTCGA CGCTGAAGGG TATGCACTGT ACTTCTCTCG CG - #CCACCATT     480    - CCTTGGGATC GTGATCGTTT TGCAGAAGGC CTTGAAACCG TTGGCGATAA CT - #TCCTGCGT     540    - CATCTTGGTA TTTATGGCTA CCGTGCAGGC TTTATCCGTC GTTACGTCAA CT - #GGCAGCCA     600    - AGTCCGTTAG AACACATCGA AATGTTAGAG CAGCTTCGTG TTCTGTGGTA CG - #GCGAAAAA     660    - ATCCATGTTG CTGTTGCTCA GGAAGTTCCT GGCACAGGTG TGGATACCCC TG - #AAGATCTC     720    - GACCCGTCGA CGAATTCTAT CGGTGGTGAC ATGAAAGACA TCTGGCGTAA CG - #AACTGTTC     780    - AAATACAAAG TTGTTCGTGT TAAACCGTTC TCTGTTGCTC CGACCCCGAT CG - #CTCGTCCG     840    - GTTATCGGTA CTGGCACCCA CCGTGAAAAA CGTGCTGTAG GTCTGGGTAT GC - #TGTTCCTG     900    - GGCGTTCTGT CTGCAGCAGG TTCCACTATG GGTGCTGCAG CTACCGCTCT GA - #CCGTACAG     960    - ACCCACTCTG TTATCAAAGG TATCGTACAG CAGCAGGACA ACCTGCTGCG TG - #CAATCCAG    1020    - GCACAGCAGG AACTGCTGCG TCTGTCTGTA TGGGGTATCC GTCAGCTGCG TG - #CTCGTCTG    1080    - CTGGCACTGG AAACCCTGAT CCAGAACCAG CAGCTGCTGA ACCTGTGGGG CT - #GCAAAGGT    1140    - CGTCTGATCT GCTACACCTC CGTTAAATGG AACGAAACCT GGCGTAACAC CA - #CCAACATC    1200    - AACCAGATCT GGGGTAACCT GACCTGGCAG GAATGGGACC AGCAGATCGA CA - #ACGTTTCT    1260    - TCCACCATCT ACGAAGAAAT CCAGAAAGCT CAGGTTCAGC AGGAACAGAA CG - #AAAAAAAA    1320    - CTGCTGGAAC TGGACGAATG GGCTTCTCTG TGGAACTGGC TGGACATCAC CA - #AATGGCTG    1380    - CGTAACATCC GTCAGGGCTA CCAGCCGCTG TCCCTGCAGA TCCCGACCCG TC - #AGCAGTCT    1440    - GAAGCTGAAA CTCCGGGTCG TACCGGTGAA GGTGGTGGTG ACGAAGGCCG TC - #CGCGTCTG    1500    - ATCCCGTCTC CGCAGGGTTT CCTGCCGCTG CTGTACACCG ACCTGCGTAC CA - #TCATCCTG    1560    - TGGTCCTACC ACCTGCTGTC TAACCTGATC TCTGGTACTC AGACTGTTAT CT - #CTCACCTG    1620    - CGTCTGGGTC TGTGGATTCT GGGTCAGAAA ATCATCGACG CTTGCCGTAT CT - #GCGCTGCT    1680    - GTTATCCACT ACTGGCTGCA GGAACTGCAG AAATCCGCTA CCTCCCTGAT CG - #ACACCTTC    1740    - GCTGTTGCAG TTGCTAACTG GACTGACGAC ATCATCCTGG GTATCCAGCG TC - #TGGGTCGT    1800    - GGTATCCTGA ACATCCCGCG TCGTGTTCGC CAGGGCTTCG AACGCTCTCT GC - #TGTAATAG    1860    - (2) INFORMATION FOR SEQ ID NO:54:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 618 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    - Met Ser Phe Val Val Ile Ile Pro Ala Arg Ty - #r Ala Ser Thr Arg Leu    #                 15    - Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly - #s Pro Met Ile Val His    #             30    - Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Gl - #u Arg Ile Ile Val Ala    #         45    - Thr Asp His Glu Asp Val Ala Arg Ala Val Gl - #u Ala Ala Gly Gly Glu    #     60    - Val Cys Met Thr Arg Ala Asp His Gln Ser Gl - #y Thr Glu Arg Leu Ala    # 80    - Glu Val Val Glu Lys Cys Ala Phe Ser Asp As - #p Thr Val Ile Val Asn    #                 95    - Val Gln Gly Asp Glu Pro Met Ile Pro Ala Th - #r Ile Ile Arg Gln Val    #           110    - Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Me - #t Thr Thr Leu Ala Val    #       125    - Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pr - #o Asn Ala Val Lys Val    #   140    - Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Ph - #e Ser Arg Ala Thr Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Le - #u Glu Thr Val Gly Asp    #               175    - Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Ty - #r Arg Ala Gly Phe Ile    #           190    - Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Le - #u Glu His Ile Glu Met    #       205    - Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Gl - #u Lys Ile His Val Ala    #   220    - Val Ala Gln Glu Val Pro Gly Thr Gly Val As - #p Thr Pro Glu Asp Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Asp Pro Ser Thr Asn Ser Ile Gly Gly Asp Me - #t Lys Asp Ile Trp Arg    #               255    - Asn Glu Leu Phe Lys Tyr Lys Val Val Arg Va - #l Lys Pro Phe Ser Val    #           270    - Ala Pro Thr Pro Ile Ala Arg Pro Val Ile Gl - #y Thr Gly Thr His Arg    #       285    - Glu Lys Arg Ala Val Gly Leu Gly Met Leu Ph - #e Leu Gly Val Leu Ser    #   300    - Ala Ala Gly Ser Thr Met Gly Ala Ala Ala Th - #r Ala Leu Thr Val Gln    305                 3 - #10                 3 - #15                 3 -    #20    - Thr His Ser Val Ile Lys Gly Ile Val Gln Gl - #n Gln Asp Asn Leu Leu    #               335    - Arg Ala Ile Gln Ala Gln Gln Glu Leu Leu Ar - #g Leu Ser Val Trp Gly    #           350    - Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Le - #u Glu Thr Leu Ile Gln    #       365    - Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Ly - #s Gly Arg Leu Ile Cys    #   380    - Tyr Thr Ser Val Lys Trp Asn Glu Thr Trp Ar - #g Asn Thr Thr Asn Ile    385                 3 - #90                 3 - #95                 4 -    #00    - Asn Gln Ile Trp Gly Asn Leu Thr Trp Gln Gl - #u Trp Asp Gln Gln Ile    #               415    - Asp Asn Val Ser Ser Thr Ile Tyr Glu Glu Il - #e Gln Lys Ala Gln Val    #           430    - Gln Gln Glu Gln Asn Glu Lys Lys Leu Leu Gl - #u Leu Asp Glu Trp Ala    #       445    - Ser Leu Trp Asn Trp Leu Asp Ile Thr Lys Tr - #p Leu Arg Asn Ile Arg    #   460    - Gln Gly Tyr Gln Pro Leu Ser Leu Gln Ile Pr - #o Thr Arg Gln Gln Ser    465                 4 - #70                 4 - #75                 4 -    #80    - Glu Ala Glu Thr Pro Gly Arg Thr Gly Glu Gl - #y Gly Gly Asp Glu Gly    #               495    - Arg Pro Arg Leu Ile Pro Ser Pro Gln Gly Ph - #e Leu Pro Leu Leu Tyr    #           510    - Thr Asp Leu Arg Thr Ile Ile Leu Trp Ser Ty - #r His Leu Leu Ser Asn    #       525    - Leu Ile Ser Gly Thr Gln Thr Val Ile Ser Hi - #s Leu Arg Leu Gly Leu    #   540    - Trp Ile Leu Gly Gln Lys Ile Ile Asp Ala Cy - #s Arg Ile Cys Ala Ala    545                 5 - #50                 5 - #55                 5 -    #60    - Val Ile His Tyr Trp Leu Gln Glu Leu Gln Ly - #s Ser Ala Thr Ser Leu    #               575    - Ile Asp Thr Phe Ala Val Ala Val Ala Asn Tr - #p Thr Asp Asp Ile Ile    #           590    - Leu Gly Ile Gln Arg Leu Gly Arg Gly Ile Le - #u Asn Ile Pro Arg Arg    #       605    - Val Arg Gln Gly Phe Glu Arg Ser Leu Leu    #   615    - (2) INFORMATION FOR SEQ ID NO:55:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 466 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    - Met Ser Phe Val Val Ile Ile Pro Ala Arg Ty - #r Ala Ser Thr Arg Leu    #                 15    - Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly - #s Pro Met Ile Val His    #             30    - Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Gl - #u Arg Ile Ile Val Ala    #         45    - Thr Asp His Glu Asp Val Ala Arg Ala Val Gl - #u Ala Ala Gly Gly Glu    #     60    - Val Cys Met Thr Arg Ala Asp His Gln Ser Gl - #y Thr Glu Arg Leu Ala    # 80    - Glu Val Val Glu Lys Cys Ala Phe Ser Asp As - #p Thr Val Ile Val Asn    #                 95    - Val Gln Gly Asp Glu Pro Met Ile Pro Ala Th - #r Ile Ile Arg Gln Val    #           110    - Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Me - #t Thr Thr Leu Ala Val    #       125    - Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pr - #o Asn Ala Val Lys Val    #   140    - Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Ph - #e Ser Arg Ala Thr Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Le - #u Glu Thr Val Gly Asp    #               175    - Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Ty - #r Arg Ala Gly Phe Ile    #           190    - Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Le - #u Glu His Ile Glu Met    #       205    - Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Gl - #u Lys Ile His Val Ala    #   220    - Val Ala Gln Glu Val Pro Gly Thr Gly Val As - #p Thr Pro Glu Asp Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Asp Pro Ser Thr Asn Ser Met Glu Gly Glu Le - #u Thr Cys Asn Ser Thr    #               255    - Val Thr Ser Ile Ile Ala Asn Ile Asp Ser As - #p Gly Asn Gln Thr Asn    #           270    - Ile Thr Phe Ser Ala Glu Val Ala Glu Leu Ty - #r Arg Leu Glu Leu Gly    #       285    - Asp Tyr Lys Leu Ile Glu Val Thr Pro Ile Gl - #y Phe Ala Pro Thr Lys    #   300    - Glu Lys Arg Tyr Ser Ser Ala Pro Val Arg As - #n Lys Arg Gly Val Phe    305                 3 - #10                 3 - #15                 3 -    #20    - Val Leu Gly Phe Leu Gly Phe Leu Ala Thr Al - #a Gly Ser Ala Met Gly    #               335    - Ala Ala Ser Leu Thr Leu Ser Ala Gln Ser Ar - #g Thr Leu Leu Ala Gly    #           350    - Ile Val Gln Gln Gln Gln Gln Leu Leu Asp Va - #l Val Lys Arg Gln Gln    #       365    - Glu Met Leu Arg Leu Thr Val Trp Gly Thr Ly - #s Asn Leu Gln Ala Arg    #   380    - Val Thr Ala Ile Glu Lys Tyr Leu Lys Asp Gl - #n Ala Gln Leu Asn Ser    385                 3 - #90                 3 - #95                 4 -    #00    - Trp Gly Cys Ala Phe Arg Gln Val Cys His Th - #r Thr Val Pro Trp Val    #               415    - Asn Asp Ser Leu Thr Pro Asp Trp Asn Asn Me - #t Thr Trp Gln Glu Trp    #           430    - Glu Lys Arg Val His Tyr Leu Glu Ala Asn Il - #e Ser Gln Ser Leu Glu    #       445    - Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Ty - #r Glu Leu Gln Lys Leu    #   460    - Asn Ser    465    - (2) INFORMATION FOR SEQ ID NO:56:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 491 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    - Met Ser Phe Val Val Ile Ile Pro Ala Arg Ty - #r Ala Ser Thr Arg Leu    #                 15    - Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly - #s Pro Met Ile Val His    #             30    - Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Gl - #u Arg Ile Ile Val Ala    #         45    - Thr Asp His Glu Asp Val Ala Arg Ala Val Gl - #u Ala Ala Gly Gly Glu    #     60    - Val Cys Met Thr Arg Ala Asp His Gln Ser Gl - #y Thr Glu Arg Leu Ala    # 80    - Glu Val Val Glu Lys Cys Ala Phe Ser Asp As - #p Thr Val Ile Val Asn    #                 95    - Val Gln Gly Asp Glu Pro Met Ile Pro Ala Th - #r Ile Ile Arg Gln Val    #           110    - Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Me - #t Ala Thr Leu Ala Val    #       125    - Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pr - #o Asn Ala Val Lys Val    #   140    - Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Ph - #e Ser Arg Ala Thr Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Le - #u Glu Thr Val Gly Asp    #               175    - Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Ty - #r Arg Ala Gly Phe Ile    #           190    - Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Le - #u Glu His Ile Glu Met    #       205    - Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Gl - #u Lys Ile His Val Ala    #   220    - Val Ala Gln Glu Val Pro Gly Thr Gly Val As - #p Thr Pro Glu Asp Pro    225                 2 - #30                 2 - #35                 2 -    #40    - Ser Thr Ala Leu Met Lys Ile Pro Gly Asp Pr - #o Gly Gly Gly Asp Met    #               255    - Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Ty - #r Lys Val Val Lys Ile    #           270    - Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ly - #s Arg Arg Val Val Gln    #       285    - Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Le - #u Phe Leu Gly Phe Leu    #   300    - Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Se - #r Met Thr Leu Thr Val    305                 3 - #10                 3 - #15                 3 -    #20    - Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gl - #n Gln Gln Asn Asn Leu    #               335    - Leu Arg Ala Ile Glu Ala Gln Gln His Leu Le - #u Gln Leu Thr Val Trp    #           350    - Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Al - #a Val Glu Arg Tyr Leu    #       365    - Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cy - #s Ser Gly Lys Leu Ile    #   380    - Cys Thr Thr Ala Val Pro Trp Asn Ala Ser Tr - #p Ser Asn Lys Ser Leu    385                 3 - #90                 3 - #95                 4 -    #00    - Glu Gln Ile Trp Asn Asn Met Thr Trp Met Gl - #u Trp Asp Arg Glu Ile    #               415    - Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Il - #e Glu Glu Ser Gln Asn    #           430    - Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Gl - #u Leu Asp Lys Trp Val    #       445    - Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Se - #r Phe Gln Thr His Leu    #   460    - Pro Ile Pro Arg Gly Pro Asp Arg Pro Glu Gl - #y Ile Glu Lys Lys Ala    465                 4 - #70                 4 - #75                 4 -    #80    - Ala Asn Val Thr Val Thr Val Pro Phe Val Tr - #p    #               490    - (2) INFORMATION FOR SEQ ID NO:57:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 651 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    - ATGATCGGTG GTGACATGAA AGACATCTGG CGTAACGAAC TGTTCAAATA CA - #AAGTTGTT      60    - CGTGTTAAAC CGTTCTCTGT TGCTCCGACC CCGATCGCTC GTCCGGTTAT CG - #GTACTGGC     120    - ACCCACCGTG AAAAACGTGC TGTAGGTCTG GGTATGCTGT TCCTGGGCGT TC - #TGTCTGCA     180    - GCAGGTTCCA CTATGGGTGC TGCAGCTACC GCTCTGACCG TACAGACCCA CT - #CTGTTATC     240    - AAAGGTATCG TACAGCAGCA GGACAACCTG CTGCGTGCAA TCCAGGCACA GC - #AGGAACTG     300    - CTGCGTCTGT CTGTATGGGG TATCCGTCAG CTGCGTGCTC GTCTGCTGGC AC - #TGGAAACC     360    - CTGATCCAGA ACCAGCAGCT GCTGAACCTG TGGGGCTGCA AAGGTCGTCT GA - #TCTGCTAC     420    - ACCTCCGTTA AATGGAACGA AACCTGGCGT AACACCACCA ACATCAACCA GA - #TCTGGGGT     480    - AACCTGACCT GGCAGGAATG GGACCAGCAG ATCGACAACG TTTCTTCCAC CA - #TCTACGAA     540    - GAAATCCAGA AAGCTCAGGT TCAGCAGGAA CAGAACGAAA AAAAACTGCT GG - #AACTGGAC     600    #            651TGTGGAA CTGGCTGGAC ATCACCAAAT GGCTGTAATA G    - (2) INFORMATION FOR SEQ ID NO:58:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 215 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    - Met Ile Gly Gly Asp Met Lys Asp Ile Trp Ar - #g Asn Glu Leu Phe Lys    #                 15    - Tyr Lys Val Val Arg Val Lys Pro Phe Ser Va - #l Ala Pro Thr Pro Ile    #             30    - Ala Arg Pro Val Ile Gly Thr Gly Thr His Ar - #g Glu Lys Arg Ala Val    #         45    - Gly Leu Gly Met Leu Phe Leu Gly Val Leu Se - #r Ala Ala Gly Ser Thr    #     60    - Met Gly Ala Ala Ala Thr Ala Leu Thr Val Gl - #n Thr His Ser Val Ile    # 80    - Lys Gly Ile Val Gln Gln Gln Asp Asn Leu Le - #u Arg Ala Ile Gln Ala    #                 95    - Gln Gln Glu Leu Leu Arg Leu Ser Val Trp Gl - #y Ile Arg Gln Leu Arg    #           110    - Ala Arg Leu Leu Ala Leu Glu Thr Leu Ile Gl - #n Asn Gln Gln Leu Leu    #       125    - Asn Leu Trp Gly Cys Lys Gly Arg Leu Ile Cy - #s Tyr Thr Ser Val Lys    #   140    - Trp Asn Glu Thr Trp Arg Asn Thr Thr Asn Il - #e Asn Gln Ile Trp Gly    145                 1 - #50                 1 - #55                 1 -    #60    - Asn Leu Thr Trp Gln Glu Trp Asp Gln Gln Il - #e Asp Asn Val Ser Ser    #               175    - Thr Ile Tyr Glu Glu Ile Gln Lys Ala Gln Va - #l Gln Gln Glu Gln Asn    #           190    - Glu Lys Lys Leu Leu Glu Leu Asp Glu Trp Al - #a Ser Leu Trp Asn Trp    #       205    - Leu Asp Ile Thr Lys Trp Leu    #   215    - (2) INFORMATION FOR SEQ ID NO:59:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1386 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    - ATGAGTTTTG TGGTCATTAT TCCCGCGCGC TACGCGTCGA CGCGTCTGCC CG - #GTAAACCA      60    - TTGGTTGATA TTAACGGCAA ACCCATGATT GTTCATGTTC TTGAACGCGC GC - #GTGAATCA     120    - GGTGCCGAGC GCATCATCGT GGCAACCGAT CATGAGGATG TTGCCCGCGC CG - #TTGAAGCC     180    - GCTGGCGGTG AAGTATGTAT GACGCGCGCC GATCATCAGT CAGGAACAGA AC - #GTCTGGCG     240    - GAAGTTGTCG AAAAATGCGC ATTCAGCGAC GACACGGTGA TCGTTAATGT GC - #AGGGTGAT     300    - GAACCGATGA TCCCTGCGAC AATCATTCGT CAGGTTGCTG ATAACCTCGC TC - #AGCGTCAG     360    - GTGGGTATGA CGACTCTGGC GGTGCCAATC CACAATGCGG AAGAAGCGTT TA - #ACCCGAAT     420    - GCGGTGAAAG TGGTTCTCGA CGCTGAAGGG TATGCACTGT ACTTCTCTCG CG - #CCACCATT     480    - CCTTGGGATC GTGATCGTTT TGCAGAAGGC CTTGAAACCG TTGGCGATAA CT - #TCCTGCGT     540    - CATCTTGGTA TTTATGGCTA CCGTGCAGGC TTTATCCGTC GTTACGTCAA CT - #GGCAGCCA     600    - AGTCCGTTAG AACACATCGA AATGTTAGAG CAGCTTCGTG TTCTGTGGTA CG - #GCGAAAAA     660    - ATCCATGTTG CTGTTGCTCA GGAAGTTCCT GGCACAGGTG TGGATACCCC TG - #AAGATCTC     720    - GACCCGTCGA CGAATTCTAT CGGTGGTGAC ATGAAAGACA TCTGGCGTAA CG - #AACTGTTC     780    - AAATACAAAG TTGTTCGTGT TAAACCGTTC TCTGTTGCTC CGACCCCGAT CG - #CTCGTCCG     840    - GTTATCGGTA CTGGCACCCA CCGTGAAAAA CGTGCTGTAG GTCTGGGTAT GC - #TGTTCCTG     900    - GGCGTTCTGT CTGCAGCAGG TTCCACTATG GGTGCTGCAG CTACCGCTCT GA - #CCGTACAG     960    - ACCCACTCTG TTATCAAAGG TATCGTACAG CAGCAGGACA ACCTGCTGCG TG - #CAATCCAG    1020    - GCACAGCAGG AACTGCTGCG TCTGTCTGTA TGGGGTATCC GTCAGCTGCG TG - #CTCGTCTG    1080    - CTGGCACTGG AAACCCTGAT CCAGAACCAG CAGCTGCTGA ACCTGTGGGG CT - #GCAAAGGT    1140    - CGTCTGATCT GCTACACCTC CGTTAAATGG AACGAAACCT GGCGTAACAC CA - #CCAACATC    1200    - AACCAGATCT GGGGTAACCT GACCTGGCAG GAATGGGACC AGCAGATCGA CA - #ACGTTTCT    1260    - TCCACCATCT ACGAAGAAAT CCAGAAAGCT CAGGTTCAGC AGGAACAGAA CG - #AAAAAAAA    1320    - CTGCTGGAAC TGGACGAATG GGCTTCTCTG TGGAACTGGC TGGACATCAC CA - #AATGGCTG    1380    #         1386    - (2) INFORMATION FOR SEQ ID NO:60:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 460 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: Protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    - Met Ser Phe Val Val Ile Ile Pro Ala Arg Ty - #r Ala Ser Thr Arg Leu    #                 15    - Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly - #s Pro Met Ile Val His    #             30    - Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Gl - #u Arg Ile Ile Val Ala    #         45    - Thr Asp His Glu Asp Val Ala Arg Ala Val Gl - #u Ala Ala Gly Gly Glu    #     60    - Val Cys Met Thr Arg Ala Asp His Gln Ser Gl - #y Thr Glu Arg Leu Ala    # 80    - Glu Val Val Glu Lys Cys Ala Phe Ser Asp As - #p Thr Val Ile Val Asn    #                 95    - Val Gln Gly Asp Glu Pro Met Ile Pro Ala Th - #r Ile Ile Arg Gln Val    #           110    - Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Me - #t Thr Thr Leu Ala Val    #       125    - Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pr - #o Asn Ala Val Lys Val    #   140    - Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Ph - #e Ser Arg Ala Thr Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Le - #u Glu Thr Val Gly Asp    #               175    - Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Ty - #r Arg Ala Gly Phe Ile    #           190    - Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Le - #u Glu His Ile Glu Met    #       205    - Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Gl - #u Lys Ile His Val Ala    #   220    - Val Ala Gln Glu Val Pro Gly Thr Gly Val As - #p Thr Pro Glu Asp Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Asp Pro Ser Thr Asn Ser Ile Gly Gly Asp Me - #t Lys Asp Ile Trp Arg    #               255    - Asn Glu Leu Phe Lys Tyr Lys Val Val Arg Va - #l Lys Pro Phe Ser Val    #           270    - Ala Pro Thr Pro Ile Ala Arg Pro Val Ile Gl - #y Thr Gly Thr His Arg    #       285    - Glu Lys Arg Ala Val Gly Leu Gly Met Leu Ph - #e Leu Gly Val Leu Ser    #   300    - Ala Ala Gly Ser Thr Met Gly Ala Ala Ala Th - #r Ala Leu Thr Val Gln    305                 3 - #10                 3 - #15                 3 -    #20    - Thr His Ser Val Ile Lys Gly Ile Val Gln Gl - #n Gln Asp Asn Leu Leu    #               335    - Arg Ala Ile Gln Ala Gln Gln Glu Leu Leu Ar - #g Leu Ser Val Trp Gly    #           350    - Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Le - #u Glu Thr Leu Ile Gln    #       365    - Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Ly - #s Gly Arg Leu Ile Cys    #   380    - Tyr Thr Ser Val Lys Trp Asn Glu Thr Trp Ar - #g Asn Thr Thr Asn Ile    385                 3 - #90                 3 - #95                 4 -    #00    - Asn Gln Ile Trp Gly Asn Leu Thr Trp Gln Gl - #u Trp Asp Gln Gln Ile    #               415    - Asp Asn Val Ser Ser Thr Ile Tyr Glu Glu Il - #e Gln Lys Ala Gln Val    #           430    - Gln Gln Glu Gln Asn Glu Lys Lys Leu Leu Gl - #u Leu Asp Glu Trp Ala    #       445    - Ser Leu Trp Asn Trp Leu Asp Ile Thr Lys Tr - #p Leu    #   460    - (2) INFORMATION FOR SEQ ID NO:61:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 873 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: Protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    - Met Ile Val Thr Met Arg Ala Met Gly Lys Ar - #g Asn Arg Lys Leu Gly    #                 15    - Ile Leu Tyr Ile Val Met Ala Leu Ile Ile Pr - #o Cys Leu Ser Ser Ser    #             30    - Gln Leu Tyr Ala Thr Val Tyr Ala Gly Val Pr - #o Val Trp Glu Asp Ala    #         45    - Ala Pro Val Leu Phe Cys Ala Ser Asp Ala As - #n Leu Thr Ser Thr Glu    #     60    - Lys His Asn Val Trp Ala Ser Gln Ala Cys Va - #l Pro Thr Asp Pro Thr    # 80    - Pro His Glu Tyr Leu Leu Thr Asn Val Thr As - #p Asn Phe Asn Ile Trp    #                 95    - Glu Asn Tyr Met Val Glu Gln Met Gln Glu As - #p Ile Ile Ser Leu Trp    #           110    - Asp Gln Ser Leu Lys Pro Cys Ile Gln Met Th - #r Phe Met Cys Ile Gln    #       125    - Met Asn Cys Thr Asp Ile Lys Asn Asn Asn Th - #r Ser Gly Thr Glu Asn    #   140    - Arg Thr Ser Ser Ser Glu Asn Pro Met Lys Th - #r Cys Glu Phe Asn Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Thr Thr Val Leu Lys Asp Lys Lys Glu Lys Ly - #s Gln Ala Leu Phe Tyr    #               175    - Val Ser Asp Leu Thr Lys Leu Ala Asp Asn As - #n Thr Thr Asn Thr Met    #           190    - Tyr Thr Leu Ile Asn Cys Asn Ser Thr Thr Il - #e Lys Gln Ala Cys Pro    #       205    - Lys Val Ser Phe Glu Pro Ile Pro Ile Tyr Ty - #r Cys Ala Pro Ala Gly    #   220    - Tyr Ala Ile Phe Lys Cys Asn Ser Ala Glu Ph - #e Asn Gly Thr Gly Lys    225                 2 - #30                 2 - #35                 2 -    #40    - Cys Ser Asn Ile Ser Val Val Thr Cys Thr Hi - #s Gly Ile Lys Pro Thr    #               255    - Val Ser Thr Gln Leu Ile Leu Asn Gly Thr Le - #u Ser Lys Glu Lys Ile    #           270    - Arg Ile Met Gly Lys Asn Ile Ser Asp Ser Gl - #y Lys Asn Ile Ile Val    #       285    - Thr Leu Ser Ser Asp Ile Glu Ile Thr Cys Va - #l Arg Pro Gly Asn Asn    #   300    - Gln Thr Val Gln Glu Met Lys Ile Gly Pro Me - #t Ala Trp Tyr Ser Met    305                 3 - #10                 3 - #15                 3 -    #20    - Ala Leu Gly Thr Gly Ser Asn Arg Ser Arg Va - #l Ala Tyr Cys Gln Tyr    #               335    - Asn Thr Thr Glu Trp Glu Lys Ala Leu Lys As - #n Thr Ala Glu Arg Tyr    #           350    - Leu Glu Leu Ile Asn Asn Thr Glu Gly Asn Th - #r Thr Met Ile Phe Asn    #       365    - Arg Ser Gln Asp Gly Ser Asp Val Glu Val Th - #r His Leu His Phe Asn    #   380    - Cys His Gly Glu Phe Phe Tyr Cys Asn Thr Se - #r Glu Met Phe Asn Tyr    385                 3 - #90                 3 - #95                 4 -    #00    - Thr Phe Leu Cys Asn Gly Thr Asn Cys Asn As - #n Thr Gln Ser Ile Asn    #               415    - Ser Ala Asn Gly Met Ile Pro Cys Lys Leu Ly - #s Gln Val Val Arg Ser    #           430    - Trp Met Arg Gly Gly Ser Gly Leu Tyr Ala Pr - #o Pro Ile Pro Gly Asn    #       445    - Leu Thr Cys Ile Ser His Ile Thr Gly Met Il - #e Leu Gln Met Asp Ala    #   460    - Pro Trp Asn Lys Thr Glu Asn Thr Phe Arg Pr - #o Ile Gly Gly Asp Met    465                 4 - #70                 4 - #75                 4 -    #80    - Lys Asp Ile Trp Arg Asn Glu Leu Phe Lys Ty - #r Lys Val Val Arg Val    #               495    - Lys Pro Phe Ser Val Ala Pro Thr Pro Ile Al - #a Arg Pro Val Ile Gly    #           510    - Thr Gly Thr His Arg Glu Lys Arg Ala Val Gl - #y Leu Gly Met Leu Phe    #       525    - Leu Gly Val Leu Ser Ala Ala Gly Ser Thr Me - #t Gly Ala Ala Ala Thr    #   540    - Ala Leu Thr Val Gln Thr His Ser Val Ile Ly - #s Gly Ile Val Gln Gln    545                 5 - #50                 5 - #55                 5 -    #60    - Gln Asp Asn Leu Leu Arg Ala Ile Gln Ala Gl - #n Gln Glu Leu Leu Arg    #               575    - Leu Ser Val Trp Gly Ile Arg Gln Leu Arg Al - #a Arg Leu Leu Ala Leu    #           590    - Glu Thr Leu Ile Gln Asn Gln Gln Leu Leu As - #n Leu Trp Gly Cys Lys    #       605    - Gly Arg Leu Ile Cys Tyr Thr Ser Val Lys Tr - #p Asn Glu Thr Trp Arg    #   620    - Asn Thr Thr Asn Ile Asn Gln Ile Trp Gly As - #n Leu Thr Trp Gln Glu    625                 6 - #30                 6 - #35                 6 -    #40    - Trp Asp Gln Gln Ile Asp Asn Val Ser Ser Th - #r Ile Tyr Glu Glu Ile    #               655    - Gln Lys Ala Gln Val Gln Gln Glu Gln Asn Gl - #u Lys Lys Leu Leu Glu    #           670    - Leu Asp Glu Trp Ala Ser Leu Trp Asn Trp Le - #u Asp Ile Thr Lys Trp    #       685    - Leu Trp Tyr Ile Lys Ile Ala Ile Ile Ile Va - #l Gly Ala Leu Ile Gly    #   700    - Val Arg Ile Val Met Ile Val Leu Asn Leu Va - #l Arg Asn Ile Arg Gln    705                 7 - #10                 7 - #15                 7 -    #20    - Gly Tyr Gln Pro Leu Ser Leu Gln Ile Pro Th - #r Arg Gln Gln Ser Glu    #               735    - Ala Glu Thr Pro Gly Arg Thr Gly Glu Gly Gl - #y Gly Asp Glu Gly Arg    #           750    - Pro Arg Leu Ile Pro Ser Pro Gln Gly Phe Le - #u Pro Leu Leu Tyr Thr    #       765    - Asp Leu Arg Thr Ile Ile Leu Trp Ser Tyr Hi - #s Leu Leu Ser Asn Leu    #   780    - Ile Ser Gly Thr Gln Thr Val Ile Ser His Le - #u Arg Leu Gly Leu Trp    785                 7 - #90                 7 - #95                 8 -    #00    - Ile Leu Gly Gln Lys Ile Ile Asp Ala Cys Ar - #g Ile Cys Ala Ala Val    #               815    - Ile His Tyr Trp Leu Gln Glu Leu Gln Lys Se - #r Ala Thr Ser Leu Ile    #           830    - Asp Thr Phe Ala Val Ala Val Ala Asn Trp Th - #r Asp Asp Ile Ile Leu    #       845    - Gly Ile Gln Arg Leu Gly Arg Gly Ile Leu As - #n Ile Pro Arg Arg Val    #   860    - Arg Gln Gly Phe Glu Arg Ser Leu Leu    865                 8 - #70    - (2) INFORMATION FOR SEQ ID NO:62:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    # 20               CATT    - (2) INFORMATION FOR SEQ ID NO:63:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    # 19               TTA    - (2) INFORMATION FOR SEQ ID NO:64:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    # 20               GGTA    - (2) INFORMATION FOR SEQ ID NO:65:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    # 19               GGT    - (2) INFORMATION FOR SEQ ID NO:66:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:    # 19               CGC    - (2) INFORMATION FOR SEQ ID NO:67:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:    #                23TCCA CAA    - (2) INFORMATION FOR SEQ ID NO:68:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:    # 20               CATC    - (2) INFORMATION FOR SEQ ID NO:69:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:    #21                TGTG G    - (2) INFORMATION FOR SEQ ID NO:70:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:    #                23GRAG AAT    - (2) INFORMATION FOR SEQ ID NO:71:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:    # 20               TAAC    - (2) INFORMATION FOR SEQ ID NO:72:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:    # 20               CARC    - (2) INFORMATION FOR SEQ ID NO:73:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:    #                 22TCA TR    - (2) INFORMATION FOR SEQ ID NO:74:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:    #                 22YCT AC    - (2) INFORMATION FOR SEQ ID NO:75:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:    #  18              AC    - (2) INFORMATION FOR SEQ ID NO:76:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:    # 20               AACC    - (2) INFORMATION FOR SEQ ID NO:77:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:    #  18              AT    - (2) INFORMATION FOR SEQ ID NO:78:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:    # 19               TCC    - (2) INFORMATION FOR SEQ ID NO:79:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:    # 20               GKTC    - (2) INFORMATION FOR SEQ ID NO:80:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:    # 20               RAGR    - (2) INFORMATION FOR SEQ ID NO:81:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:    #                 22TTC TT    - (2) INFORMATION FOR SEQ ID NO:82:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:    #                 22GTT AA    - (2) INFORMATION FOR SEQ ID NO:83:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:    # 19               CAC    - (2) INFORMATION FOR SEQ ID NO:84:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:    #21                ATGT C    - (2) INFORMATION FOR SEQ ID NO:85:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:    # 20               ACTT    - (2) INFORMATION FOR SEQ ID NO:86:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:    # 19               TGG    - (2) INFORMATION FOR SEQ ID NO:87:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:    # 19               GGG    - (2) INFORMATION FOR SEQ ID NO:88:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:    #          32      AGGG GAGTTGACCT GC    - (2) INFORMATION FOR SEQ ID NO:89:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 34 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:    #        34        GCTA TTTAGTTTTT GTAG    __________________________________________________________________________

We claim:
 1. A method for simultaneously detecting and differentiatingbetween analytes comprising antibodies to HIV-1 group O, HIV-1 group M,and HIV-2 in a test sample, comprising:(a) contacting said test samplewith an analytical device having a strip with a proximal end and adistal end, wherein said test sample moves from said proximal end toabout said distal end by capillary action, and wherein said stripcontains at least one immobilized capture reagent per analyte, for atime and under conditions sufficient to form capture reagent / analytecomplexes by the binding of said analyte and said capture reagent; and(b) determining the presence of the analyte(s) by detecting a visiblecolor change at the capture reagent site on the strip,wherein saidcapture reagent for HIV-1 group O comprises a polypeptide selected fromthe group consisting of SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60, said capture reagent forHIV-1 group M comprises a polypeptide SEQ ID NO: 56, and said capturereagent for HIV-2 comprises a polypeptide SEQ ID NO:
 55. 2. The methodof claim 1, wherein said immobilized capture reagent is configured as aletter, number, icon, or symbol.
 3. The method of claim 1, wherein alabeled reagent is contained within the strip in a situs between theproximal end and the immobilized patient capture reagent.
 4. The methodof claim 1, wherein said polypeptide capture reagents are produced byrecombinant technology.
 5. The method of claim 3, wherein said labeledreagent is selenium.
 6. The method of claim 1, wherein said test sampleis a body fluid.
 7. The method of claim 6, wherein said body fluid isselected from the group consisting of whole blood, serum, plasma, urineand saliva.
 8. An analytical device for simultaneous detecting anddifferentiating between HIV-1 group O, HIV-1 group M and HIV-2 in a testsample, comprising a strip with a proximal end and a distal end, whereinsaid test sample is capable of moving from said proximal end to aboutsaid distal end by capillary action, and wherein said strip contains atleast one immobilized capture reagent per analyte, for binding of saidanalyte and said capture reagent; and wherein said capture reagent forHIV-1 group O comprises a polypeptide selected from the group consistingof SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ IDNO: 58, and SEQ ID NO: 60, said capture reagent for HIV-1 group Mcomprises a polypeptide SEQ ID NO: 56, and said capture reagent forHIV-2 comprises a polypeptide SEQ ID NO:
 55. 9. The analytical device ofclaim 8, wherein said immobilized capture reagent is configured as aletter, number, icon, or symbol.
 10. The analytical device of claim 8,wherein a labeled reagent is contained within the strip in a situsbetween the proximal end and the immobilized patient capture reagent.11. The analytical device of claim 10, wherein said labeled reagent isselenium.
 12. The analytical device of claim 8, wherein said test sampleis a body fluid.
 13. The analytical device of claim 12, wherein saidbody fluid is selected from the group consisting of whole blood, serum,plasma, urine and saliva.
 14. The analytical device of claim 8 whereinsaid polypeptide capture reagents are produced by recombinanttechnology.
 15. A kit for use in specific binding assays, having ananalytical device for determining the presence or amount of HIV-1 groupO, HIV-1 group M and HIV-2 in a test sample, comprising a strip having aproximal end and a distal end, wherein said test sample is capable ofmoving from said proximal end to about said distal end by capillaryaction, and wherein said strip contains an immobilized capture reagentthat binds to a member selected from the group consisting of theanalyte, an ancillary specific binding member and a labeled reagent, andwherein said capture reagent for HIV-1 group O comprises a polypeptideselected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 50, SEQID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60, said capturereagent for HIV-1 group M comprises a polypeptide SEQ ID NO: 56, andsaid capture reagent for HIV-2 comprises a polypeptide SEQ ID NO: 55.16. The test kit of claim 15 wherein said labeled reagent is selenium.17. The test kit of claim 15, further comprising a positive reagentcontrol.
 18. The test kit of claim 15, further comprising a negativereagent control.
 19. The test kit of claim 15, wherein said polypeptidecapture reagents are produced by recombinant technology.